Methods of reducing adverse effects of non-thermal ablation

ABSTRACT

The present invention provides systems, methods, and devices for electroporation-based therapies (EBTs). Embodiments provide patient-specific treatment protocols derived by the numerical modeling of 3D reconstructions of target tissue from images taken of the tissue, and optionally accounting for one or more of physical constraints or dynamic tissue properties. The present invention further relates to systems, methods, and devices for delivering bipolar electric pulses for irreversible electroporation exhibiting reduced or no damage to tissue typically associated with an EBT-induced excessive charge delivered to the tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 14/808,679, filed Jul. 24, 2015. The '679application is a Divisional application of U.S. patent application Ser.No. 12/906,923, filed Oct. 18, 2010, which issued as U.S. Pat. No.9,198,733 on Dec. 1, 2015, and which parent application claims priorityto and the benefit of the filing date of U.S. Provisional ApplicationNo. 61/252,445, filed Oct. 16, 2009. The '923 application is aContinuation-in-Part (CIP) of U.S. patent application Ser. No.12/757,901, filed Apr. 9, 2010, which issued as U.S. Pat. No. 8,926,606on Jan. 6, 2015, and which claims priority to U.S. ProvisionalApplication Nos. 61/167,997, filed Apr. 9, 2009, and 61/285,618, filedDec. 11, 2009; and the '923 application is a CIP of U.S. patentapplication Ser. No. 12/609,779, which was filed Oct. 30, 2009, andwhich issued as U.S. Pat. No. 8,465,484 on Jun. 18, 2013. The '923application is a CIP of U.S. application Ser. No. 12/491,151, filed Jun.24, 2009, which issued as U.S. Pat. No. 8,992,517 on Mar. 31, 2015, andwhich claims priority to U.S. Provisional Application Nos. 61/075,216,filed Jun. 24, 2008, 61/171,564, filed Apr. 22, 2009, and 61/167,997,filed Apr. 9, 2009, and the '151 application is a CIP of U.S. patentapplication Ser. No. 12/432,295, which was filed Apr. 29, 2009, andwhich issued as U.S. Pat. No. 9,598,691 on Mar. 21, 2017, and whichclaims priority to U.S. Provisional Application No. 61/125,840, filedApr. 29, 2008, each of which is hereby incorporated by reference hereinin its entirety. Further, the '679 application is a Continuation-in-Partapplication of U.S. patent application Ser. No. 13/332,133, filed Dec.20, 2011, which issued as U.S. Pat. No. 10,448,989 on Oct. 22, 2019. The'133 application is a Continuation-in-Part application of U.S. patentapplication Ser. No. 12/757,901, filed Apr. 9, 2010, which issued asU.S. Pat. No. 8,926,606 on Jan. 6, 2015. The '901 application relies onthe disclosure of and claims priority to and the benefit of the filingdate of U.S. Provisional Application Nos. 61/167,997, filed Apr. 9,2009, and 61/285,618, filed Dec. 11, 2009. Additionally, the '133application relies on the disclosure of and claims priority to and thebenefit of the filing date of U.S. Provisional Application No.61/424,872, filed Dec. 20, 2010.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention provides systems, methods, and devices forelectroporation-based therapies (EBTs). Embodiments providepatient-specific treatment protocols derived by the numerical modelingof 3D reconstructions of target tissue from images taken of the tissue,and optionally accounting for one or more of physical constraints and/ordynamic tissue properties. The present invention further relates tosystems, methods, and devices for delivering bipolar electric pulses forirreversible electroporation without damage to tissue typicallyassociated with an EBT-induced excessive charge delivered to the tissueand mitigate electrochemical effects that may distort the treatmentregion.

Description of Related Art

Irreversible electroporation (IRE) and other electroporation-basedtherapies (EBTs), such as electrogenetransfer or electrochemotherapy,may often be administered in a minimally invasive fashion. There are,however, several considerations that may lead to an increase in thedifficulty of administering such treatments. This includes typicalapplications where deep targeted regions are treated by placing needleor other electrodes deep into the tissue, where one can no longerdirectly visualize the affected area. There is some evidence thatchanges in the tissue's permeability, and therefore also its electricalconductivity, allow one to visualize and monitor affected regions inreal-time. These changes are most pronounced in homogeneous andimage-dense tissues, such as hyperechoic ultrasound tissues, whereincreased permeability decreases the electroporated echogenicity.However, many tumors and other tissues are far too heterogeneous orexhibit properties that do not allow for simple visualization of theelectroporated areas. In addition, these changes for real-time imagingtypically only designate electroporated regions, not necessarily thosekilled for IRE therapies.

In applying EBTs, ensuring adequate coverage of the targeted region(e.g., any mass or lesion or undesirable tissue to be affected,including margins beyond the lesion itself), while sparing healthytissues is vital to therapeutic success. Due to the limitations inherentin treating deep tissues without exposing them, it is critical forpractitioners to develop and implement treatment protocols capable ofachieving their clinical objectives.

Furthermore, typical electrodes and pulsing parameters (number ofpulses, pulse polarity, pulse length, repetition rate, pulse shape,applied voltage, electrode geometry and orientation, etc.) will have alarge impact on the affected areas. Typical therapeutic geometriesdictated by current electrode setups will be ellipsoidal in generalshape. However, many tumors do not distinctly fit the shapes created bya single setup of an electrode. Therefore, successful implementation ofEBTs typically requires a complex array of electrodes and pulseparameters arranged in a specific manner to ensure complete treatment ofthe targeted area while minimizing effects to healthy tissue and sparingvital structures. Such predictions of superimposing treatment regionsfor complex protocols can be cumbersome. Therefore, treatment planningtechniques that aid or allow a practitioner to develop general treatmentprotocols for most clinical tumors are typically used to effectivelycapitalize on the great therapeutic potential for IRE and other EBTs.

Current treatment planning techniques from systems such as theNanoKnife® utilize interpolations and analytical techniques to aidpractitioner treatment region predictions. The interpolation techniquesprovide the physician with diagrams of 3D numerical model solutionpredicted treatment areas from very specific settings, including anexact number of pulses, pulse length, voltage, and electrode setup(e.g., separation distance, exposure length, and diameter) withdimensions provided for the treatment areas in 2 planes and the generalshape. The predicted treatment dimensions are taken from theexperimental results of applying that specific set of conditions inexperimental subjects, typically in healthy, homogeneous environments.It is from this diagram of expected region, that the physician would setup their electrodes the same way and use the same pulses and arrangemultiple applications to the point where they anticipate they will havetreated the entire volume.

There is room, however, for improvement in such systems. If the targetedvolume is smaller than the dimensions in the diagram, the practitionerhas no information about how much to change the physical setup (exposurelength, separation distance, etc.), or pulse parameters (voltage, numberof pulses, etc.) in order to prevent damaging the surrounding tissue. Inanother example, if the shape does not fit that of the diagram, thepractitioner will not be able to adjust the protocol to minimize damagebeyond the targeted margin while still treating the targeted area.

In another solution to facilitating practitioner treatment planning,software is provided that uses a lookup table of treatment dimensions oruses a calibrated analytical solution to mimic the shape of numericalsimulations. The lookup table may be taken from a large compilation ofsimulations run at varying physical and pulse parameters, wheredimensions of interest for predicted treatment regions are taken basedon a calibrated electric field threshold found to represent the affectedmargin of interest observed in experiments on healthy tissue (IRE,reversible electroporation, no electroporation, thermal damage).

Although the lookup table would allow a practitioner to manipulate theabove variables and receive real-time feedback on predicted dimensions,the geometry of the affected region is often more complex than can besummarized with a few dimensions. Therefore, analytical solutions forthe shape of the electric field distribution have been developed and arethe current state-of-the-art on the NanoKnife® system. These solutionsare able to mimic the shape of the electric field distribution fromtypical numerical simulations. The value of electric field contour isthen matched to that seen from the numerical solution so that they bothrespond to their physical and pulse conditions in approximately the samemanner. A calibration can then be used to adjust the size, and thereforevarious electric field thresholds (IRE, reversible, no electroporation,thermal damage) depicted to provide predicted affected regions. Thepractitioner may then adjust the variables such as voltage andseparation distance (currently the only two that account for changes inpredicted margins in the NanoKnife® embodiment), and see how thepredicted affected margins vary in real-time. This provides thepractitioner a much better method to find and place an appropriateelectrode array with variable voltages to treat the entire region. Thereis also an optimization autoset probes function that places the probesand sets the voltage based on the number of probes selected and threedimensions input for the targeted region (assuming it to be a perfectellipsoid).

The current state-of-the-art provides a very basic, fundamentalexplanation to practitioners about predicted treatment regions.Application of the current techniques in real-life clinical andexperimental scenarios in which EBTs will typically be used provides tothe practitioner helpful but inflexible tools.

For example, the analytical embodiment is a simple cross-sectional viewof predicted margins at the center of the electrodes. This means that itcannot account for the falloff of electric field distribution (andtherefore affected margins) at the tips of the electrodes. Although useof this approach can mimic the shape and size of these regions in 2D, itis not possible to accurately depict 3D scenario shapes in detail.Further, the lookup table cannot easily provide an accurate 3D shape,nor can the analytical solution be adapted.

True electroporation applications will increase the conductivity of theaffected regions, which will in turn change the size and shape of theelectric field distribution. A comparison of the electric fielddistribution (A,C) and conductivity map (B,D) of two identical numericalmodels without (A,B) and with (C,D) changing conductivity is shown inFIGS. 1A-D. From these figures, one can see how the conductivityincreases from 0.1 S/m (the baseline level for the entire tissue domain,constant in part B) up to 0.155 S/m, an increase of 55%, for regionsexperiencing predicted IRE (deep red in part D), with regionsexperiencing varying extents of predicted reversible electroporationfilling in between this (cyan through bright red). This change inconductivity in response to electroporation effects results in analtered electric field distribution, which may be seen in part C, wherethe distribution is larger, especially at the region between theelectrodes. Changes in conductivity have been observed to reach severaltimes higher than the baseline conductivity in the literature. Thesechanges can be simulated in numerical solutions, and the general sizechanges can be accounted for with some accuracy in the analyticalsolutions by recalibrating them, but their shape is fixed, and cannotaccurately reflect the predicted affected region's shape whenconsidering changing conductivity.

Tumors will often have different electrical and physical properties thantheir neighboring tissues or even from their native tissues of origin(e.g., cancerous astrocytes which may not behave the same as normalones). In addition, surrounding tissues of different tissue types willalso have different properties from each other (bone, muscle, fat,blood). These differences in electrical properties will alter theelectric field distribution for a given application of EBTs. Because theelectric field to which the tissue is exposed is the primary determinantin the effect on the cell, these changes will change the shape and sizeof the affected regions. Numerical simulations are capable of modelingthe electric field distribution in such heterogeneous systems. However,the rigid analytical solutions cannot be adjusted to account for suchdifferences, and therefore could not as accurately predict affectedregions for the different environments in clinical cases. The analyticalsolution, e.g., could not predict the differences between a tumorsituated adjacent to the skull, the quadriceps muscle, or the heart.Although lookup tables could theoretically be developed for thedimensions of the affected regions in a number of environments, thegreat variability between the anatomy of each patient, each specifictumor, and each exact tumor location relative to its environment isimpractical and futile.

FIGS. 2A-J demonstrate the effect of heterogeneous systems on electricfield distribution. These figures show the electric field andtemperature distribution for a three-dimensional numerical model. Moreparticularly, FIG. 2J shows the model setup, where two needle electrodes(1 mm in diameter) are placed within the outer borders of a targetedregion of tissue, surrounded by a peripheral region. The red and blackregions on the electrodes represent the energized surfaces, where 4200 Vwas applied to one electrode and the other was set to ground. Thethermal properties were set to represent a targeted region of a tumorwithin fat. The electrical conductivity for the targeted (σ_(r)) andperipheral (σ_(p)) tissues was manipulated between 0.025 and 0.25 S/m toestablish conductivity ratios (σ_(t)/σ_(p); relative conductivities ofthe targeted/peripheral region) of 0.1, 1, and 10. FIGS. 2A-I show thenumerical model outputs for conductivity ratios (σ_(t)/σ_(p)) of 0.1(A,D,G), 1 (B,E,H), and 10 (C,F,I); showing electric field (A-F) duringthe pulse and temperature (G-I) distributions 1 second after the firstpulse. The higher conductivity ratios show progressively more areatreated by IRE with less thermal effects. Targeted tissue boundary maybe seen as the solid black line. Observing the electric fielddistribution at the boundary shows that the shape is also changing (notjust size) as a result of the heterogeneous environment. Existingtreatment planning systems are not capable of accounting for suchdynamic tissue properties in real time.

The current embodiment of the treatment planning software still leavesit up to the practitioner to select a desired number of probes, butprovides no simple method of showing how the optimized distributionswill be shaped if the user wants to directly compare using differentnumbers of probes for a given lesion. The current system therefore alsodoes not select the optimal number of probes for the user, a questionthat may be difficult to answer for more complex electrode geometries.

Temperature changes associated with Joule-type resistive heating of thetissue will also affect local regions conductivity based on itstemperature (typically increases by approximately 3%/° C.). This willalso change the size and shape of the electric field distribution basedon the parameters used; including the number of pulses, pulse length,and repetition rate for an entire protocol (more pulses of longer lengthwith higher repetition rates will all increase the thermally-associatedconductivity changes, increasing this variation). Because the currenttreatment planning tools are based on simulations from the electricfield distribution of a single application of a pulse, these dynamicconductivity behaviors also cannot be taken into account. Something thatdoes would have to be able to simulate the changes that occur as aresult of thermal effects on conductivity.

The current state of the art does allow the practitioner to describe thesize/shape of the lesion in very basic dimensional terms (length, width,depth). This shape is then superimposed to scale with the predictedtreatment regions, allowing a practitioner to ensure appropriatedistribution and coverage. Although we have already pointed out theinsufficiencies in handling this third dimension, it should also bepointed out that the basic ellipsoidal shape assumed by this system iswholly inadequate at describing the complex, often irregular, asymmetricgeometries that tumors may take in clinical settings. The practitioneris thus left currently with assessing treatment protocol adequacy in 2Dterms.

What is needed is a technique and system (or a series of independentsystems) that allows a practitioner to accurately plan and implement inreal time patient-specific treatment protocols which are capable ofaccounting for dynamic tissue properties and which can be used withaccuracy and reliability in the clinical or experimental setting forEBTs.

SUMMARY OF THE INVENTION

The numerous limitations inherent in the planning system described aboveprovide great incentive for a new, better system capable of accountingfor one or more of these issues. If EBTs are to be seen as an accurate,reliable therapeutic method, then treatment planning methods andpackages should be developed that can more accurately predict treatmentoutcomes with these considerations taken into account in apatient-to-patient basis.

The primary limitation to the above-mentioned, state-of-the-arttreatment planning system is its need to provide treatment predictionsin real-time, where a practitioner would be capable of changing thevoltage or geometry parameters of a treatment protocol and immediatelysee how that impacts the entire treatment region. However, as morecomplex tumor shapes, sizes, and environments are encountered, real-timeevaluation of superimposed treatment regions is cumbersome at best andinadequate to develop reliable therapies. Therefore, a more advancedsystem that allows treatment planning in advance of applying the therapywould be ideal to handling these detailed procedures. This allows forthe adaptation of numerical solutions to provide treatment regions.

Accordingly, embodiments of the invention provide treatment planningsystems, methods, and devices for determining a patient-specificelectroporation-based treatment protocol comprising: a) a moduleoperably configured to receive and process information from medicalimages of a target structure to prepare a 3-D reconstruction model ofthe target structure; and b) a module operably configured to perform anumerical model analysis using as inputs in the analysis the 3-Dreconstruction and information from one or more of physical constraints,tissue heterogeneities, dynamic effects of electropermeabilization,dynamic thermal effects, or effects resulting from multiple treatments;and c) a module operably configured to construct one or more electricalprotocols defining a treatment region and treatment parameters foreffectively treating the target structure.

Further included in embodiments of the invention are treatment planningsystems for determining a patient-specific electroporation-basedtreatment protocol comprising: a) a processing module operablyconfigured for performing the following stages: 1) receiving andprocessing information from medical images of a target structure andpreparing a 3-D reconstruction model of the target structure; 2)performing a numerical model analysis using as inputs in the analysisthe 3-D reconstruction and information from one or more of physicalconstraints, tissue heterogeneities, dynamic effects ofelectropermeabilization, dynamic thermal effects, or effects resultingfrom multiple treatments; and 3) constructing one or more protocols eachproviding a treatment region with parameters for electroporating thetarget structure; and b) a processor for executing the stages of theprocessing module.

Such treatment planning systems can comprise a processing module capableof performing one or more of the stages in real time.

Information from medical images to be analyzed in treatment systemsaccording to embodiments of the invention can be extracted from one oran array of images obtained from pathologic specimens or one or moreimaging modalities chosen from radiographs, tomograms, nuclearscintigraphic scans, CT, MRI, PET, or US. The information from one ormore of these sources can be compiled to prepare a 3D reconstruction ofthe target area, which is represented by a surface or a solid volume.The treatment planning systems according to embodiments of the inventioncan have as a target structure a) a targeted region or mass; orb) atargeted region or mass with neighboring regions; or c) a 3D map ofvoxels to be treated as independent elements in the finite modelingsoftware.

Preferred numerical model analysis for treatment systems of theinvention comprise finite element modeling (FEM). Even more preferred astreatment planning systems, wherein the numerical model analysisinvolves accounting for physical constraints, tissue heterogeneities,dynamic effects of electropermeabilization, dynamic thermal effects, andmultiple treatment effects.

Even further, self-optimization algorithms for constructing thetreatment protocols can also be incorporated into the inventive methods,systems, and devices. For example, the treatment planning systems cancomprise a self-optimization algorithm which is capable of repeatedlyevaluating one or more of physical constraints, placement of electrodes,electric field distribution simulations, and evaluation of outcomesuccess until one or more effective protocol is constructed. It can alsogenerate a predicted treatment time that will aid the physician indetermining the optimal protocol.

According to some embodiments of the invention, the treatment planningsystems can involve automatically, interactively, or automatically andinteractively with or without user input determining the treatmentregion and parameters for electroporating.

Such treatment planning systems can also be capable of constructingprotocols for an initial patient treatment or retreatment with orwithout additional medical images.

Treatment systems according to embodiments of the invention can also beadapted to instruct an electrical waveform generator to perform theprotocol.

Such systems can further comprise an electrical waveform generator inoperable communication with the processing module and capable ofreceiving and executing the treatment protocol.

Instructions for implementing the treatment protocols can comprisespecifying a number of bipolar pulses to be delivered, a length of pulseduration, and a length of any delay between pulses. Additionally, thegenerators of such treatment systems can be operably configured fordelivering a bipolar pulse train.

Methods and devices incorporating one or more of the features of thetreatment planning systems according to the invention are alsoconsidered embodiments.

In particular, treatment planning methods can comprise: a) receiving andprocessing information from medical images of a target structure andpreparing a 3-D reconstruction model of the target structure; b)performing a numerical model analysis using as inputs in the analysisthe 3-D reconstruction and information from one or more of physicalconstraints, tissue heterogeneities, dynamic effects ofelectropermeabilization, dynamic thermal effects, or effects resultingfrom multiple treatments; and c) constructing an electroporationprotocol based on results of the analyzing; wherein the receiving,processing, analyzing, and constructing is performed in real time.

Other methods may comprise method steps for reducing adverse effects ofirreversible electroporation of tissue comprising administeringelectrical pulses through electrodes to tissue in a manner which causesirreversible electroporation of the tissue but minimizes electricalcharge build up on the electrodes, or minimizes charge delivered to thetissue, or both. Adverse effects to be avoided may include, to name afew, one or more of thermal damage of the tissue, deleteriouselectrochemical effects, or electrolysis.

Preferred methods according to the invention may comprise electricalpulses comprising a series of unipolar and bipolar pulses with a netcharge of zero. More particularly, the net charge of zero can beachieved by a change in potential direction for each pulse, or a changein potential direction within each pulse.

Further, electrical pulses generated in the methods can togethercomprise a pulse protocol comprising a train of unipolar pulses followedby a train of unipolar pulses of opposite polarity, or a train ofbipolar pulses, or simultaneous unipolar pulses of opposite polaritywhich are offset from one another by a desired amount, or a combinationof protocols.

Electrical pulses used in the methods, systems, and devices of theinvention can have a waveform which is square, triangular, trapezoidal,exponential decay, sawtooth, sinusoidal, or of alternating polarity, orcomprise a combination of one or more waveforms.

Control systems for electroporation devices are also consideredembodiments of the present invention. Such systems can be configured tocomprise: a) a processor in operable communication with a controlmodule; b) a control module executable by the processor and in operablecommunication with an electrical circuit, wherein the control module isoperably configured for initiating switching of the circuit at a rate ofbetween 10 ms to 1 ns; and c) an electrical circuit operably configuredto enable delivery of a voltage to an electrode and switching of thevoltage to a second electrode to cause reversing of the polarity of theelectric potential between the two electrodes.

Similarly, electroporation system embodiments of the invention cancomprise: a) an electroporation device capable of delivering a firstunipolar electrical pulse; b) the electroporation device further capableof, or a second electroporation device capable of, delivering a secondunipolar electrical pulse which is opposite in polarity to the firstunipolar pulse; c) a processor in operable communication with a controlmodule; d) a control module executable by the processor and in operablecommunication with the electroporation device(s), wherein the controlmodule is operably configured for initiating delivery of the firstunipolar electrical pulse at a time 1 and for initiating delivery of thesecond unipolar electrical pulse at time 2 offset from time 1 by 1second to 1 nanosecond.

Electroporation devices can also be operably configured to enabledelivery of an electrical pulse to a first electrode, switching of thepulse to a second electrode to cause reversing of the polarity of theelectric potential between the two electrodes, and switching of thepulse back to the first electrode or to zero, wherein a cycle ofswitching is established which cycle is capable of being performed at arate of between 10 milliseconds to 1 nanosecond.

Such devices, systems, and methods can be configured to provide forswitching to occur between or within the electrical pulse. Devices, forexample, can be configured such that the electrical pulses togethercomprise a pulse protocol comprising a train of unipolar pulses followedby a train of unipolar pulses of opposite polarity or a train of bipolarpulses.

Aspects of the invention include Aspect 1, a treatment planning systemfor determining a patient-specific electroporation-based treatmentprotocol comprising: a processing module operably configured forperforming the following stages: receiving and processing informationfrom medical images of a target structure and preparing a 3-Dreconstruction model of the target structure; performing a numericalmodel analysis using as inputs in the analysis the 3-D reconstructionand information from one or more of physical constraints, tissueheterogeneities, dynamic effects of electropermeabilization, dynamicthermal effects, or effects resulting from multiple treatments; andconstructing one or more protocols each providing a treatment regionwith parameters for electroporating the target structure; and aprocessor for executing the stages of the processing module.

Aspect 2 is the treatment planning system of Aspect 1, wherein theprocessing module is capable of performing the stages in real time.

Aspect 3 is the treatment planning system of Aspect 1, wherein theinformation from medical images is extracted from an array of imagesobtained from one or more imaging modalities chosen from radiographs,tomography, nuclear scintigraphy, CT, MRI, fMRI, PET, or US.

Aspect 4 is the treatment planning system of Aspect 1, wherein thenumerical model analysis comprises finite element modeling (FEM).

Aspect 5 is the treatment planning system of Aspect 1, wherein the 3Dreconstruction is a surface or a solid volume.

Aspect 6 is the treatment planning system of Aspect 4, wherein thetarget structure is a targeted region or mass; or is a targeted regionor mass with neighboring regions; or is a 3D map of voxels to be treatedas independent elements in the finite modeling software.

Aspect 7 is the treatment planning system of Aspect 1, wherein thenumerical model analysis involves accounting for physical constraints,tissue heterogeneities, dynamic effects of electropermeabilization,dynamic thermal effects, and multiple-treatment effects.

Aspect 8 is the treatment planning system of Aspect 1, furthercomprising a self-optimization algorithm for constructing the protocols.

Aspect 9 is the treatment planning system of Aspect 8, wherein theself-optimization algorithm is capable of repeatedly evaluating one ormore of physical constraints, placement of electrodes, electric fielddistribution simulations, and outcome success of the, and evaluation ofoutcome success until one or more effective protocol is constructed.

Aspect 10 is the treatment planning system of Aspect 1, wherein thetreatment region and parameters for electroporating are determinedautomatically, interactively, or automatically and interactively with orwithout user input.

Aspect 11 is the treatment planning system of Aspect 1, capable ofconstructing protocols for an initial patient treatment or retreatmentwith or without additional medical images.

Aspect 12 is the treatment planning system of Aspect 1, further adaptedfor instructing an electrical waveform generator to perform theprotocol.

Aspect 13 is the treatment planning system of Aspect 12, furthercomprising an electrical waveform generator in operable communicationwith the processing module and capable of receiving and executing thetreatment protocol.

Aspect 14 is the treatment planning system of Aspect 12, whereininstructing comprises specifying a number of bipolar pulses to bedelivered, a length of pulse duration at any pole, and a length of anydelay between pulses.

Aspect 15 is the treatment planning system of Aspect 13, wherein thegenerator is operably configured for delivering a bipolar pulse train.

Aspect 16 is the treatment planning system of Aspect 2, wherein theprocessing module further comprises functionality for monitoringelectrode or tissue temperature in real time and for consideringelectrode or tissue temperature in the analysis.

Aspect 17 is a treatment planning method comprising: receiving andprocessing information from medical images of a target structure andpreparing a 3-D reconstruction model of the target structure; performinga numerical model analysis using as inputs in the analysis the 3-Dreconstruction and information from one or more of physical constraints,tissue heterogeneities, dynamic effects of electropermeabilization,dynamic thermal effects, or effects resulting from multiple treatments;and constructing an electroporation protocol based on results of theanalyzing; wherein the receiving, processing, analyzing, andconstructing is performed in real time.

Aspect 18 is a method of reducing adverse effects of irreversibleelectroporation comprising administering electrical pulses throughelectrodes to tissue in a manner which causes irreversibleelectroporation of the tissue but minimizes electrical charge build upon the electrodes, or minimizes charge delivered to the tissue, or both.

Aspect 19 is the method of Aspect 18, wherein the adverse effects areone or more of thermal damage of the tissue or electrolysis.

Aspect 20 is the method of Aspect 19, wherein the electrical pulsescomprise a series of unipolar or bipolar pulses with a net charge ofzero.

Aspect 21 is the method of Aspect 20, wherein the net charge of zero isachieved by a change in potential direction between each pulse, or achange in potential direction within each pulse.

Aspect 22 is the method of Aspect 20, wherein the electrical pulsestogether comprise a pulse protocol comprising a train of unipolar pulsesfollowed by a train of unipolar pulses of opposite polarity, or a trainof bipolar pulses, or simultaneous unipolar pulses of opposite polaritywhich are offset from one another by a desired amount, or a combinationof protocols.

Aspect 23 is the method of Aspect 22, wherein the electrical pulses havea waveform which is square, triangular, trapezoidal, exponential decay,sawtooth, sinusoidal, or of alternating polarity, or comprise acombination of one or more waveform.

Aspect 24 is a control system for an electroporation device comprising:a processor in operable communication with a control module; a controlmodule executable by the processor and in operable communication with anelectrical circuit, wherein the control module is operably configuredfor initiating switching of the circuit at a rate of between 10 ms to 1ns; and an electrical circuit operably configured to enable delivery ofa voltage to an electrode and switching of the voltage to a secondelectrode to cause reversing of the polarity of the electric potentialbetween the two electrodes.

Aspect 25 is a electroporation system comprising: an electroporationdevice capable of delivering a first unipolar electrical pulse; theelectroporation device further capable of, or a second electroporationdevice capable of, delivering a second unipolar electrical pulse whichis opposite in polarity to the first unipolar pulse; a processor inoperable communication with a control module; a control moduleexecutable by the processor and in operable communication with theelectroporation device(s), wherein the control module is operablyconfigured for initiating delivery of the first unipolar electricalpulse at a time 1 and for initiating delivery of the second unipolarelectrical pulse at time 2 offset from time 1 by 1 second to 1nanosecond.

Aspect 26 is an electroporation device operably configured to enabledelivery of an electrical pulse to a first electrode, switching of thepulse to a second electrode to cause reversing of the polarity of theelectric potential between the two electrodes, and switching of thepulse back to the first electrode or to zero, wherein a cycle ofswitching is established which cycle is capable of being performed at arate of between 10 milliseconds to 1 nanosecond.

Aspect 27 is the device of Aspect 26, wherein the switching occurs foreach electrical pulse or within each electrical pulse.

Aspect 28 is the device of Aspect 27, wherein the electrical pulsestogether comprise a pulse protocol comprising a train of unipolar pulsesfollowed by a train of unipolar pulses of opposite polarity or a trainof bipolar pulses.

Aspect 29 is the device of Aspect 28, wherein the electrical pulses havea waveform which is square, triangular, trapezoidal, exponential decay,sawtooth, sinusoidal, or of alternating polarity, or comprise acombination of one or more waveform.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are schematic diagrams comparing the electric fielddistribution (A,C) and conductivity map (B,D) of two identical numericalmodels without (A,B) and with (C,D) changing conductivity.

FIGS. 2A-I are schematic diagrams showing the numerical model outputsfor conductivity ratios (σ_(t)/σ_(p)) of 0.1 (A,D,G), 1 (B,E,H), and 10(C,F,I); showing electric field (A-F) during the pulse and temperature(G-I) distributions 1 second after the first pulse.

FIG. 2J is a schematic diagram showing placement of the electrodes inthe targeted tissue for the set up illustrated in FIGS. 2A-I.

FIG. 3 is a series of CT images showing the presence of a tumor in theleft thigh of the canine patient of Example I.

FIG. 4 is a CT image from FIG. 3 , within which the region of interestis traced.

FIG. 5 is a drawing of a 3D reconstruction of the target region ofExample I, which was reconstructed by compiling a series of axial tracesto create a representative shape of the targeted region in threedimensions.

FIG. 6 is the drawing of the 3D reconstructed geometry shown in FIG. 5visualized relative to the rest of the patient.

FIG. 7 is a graphic representation of the 3D reconstruction of FIG. 5 asimported into and converted within Comsol Multiphysics.

FIG. 8 is a graph from Duck, 1990, showing the relationship betweenconductivity and %-water, which may also be used to estimate a tissue'selectrical properties.

FIG. 9 is the drawing of the 3D reconstruction of the target tumor ofFIG. 5 visualized in relation to surrounding structures within the body,which is a tool useful for developing treatment constraints.

FIG. 10 is a graphic representation of the 3D reconstruction of FIG. 5as imported into and converted within Comsol Multiphysics and furtherincluding a demonstrative electrode placement for an exemplary treatmentprotocol.

FIG. 11A is a schematic representation of an electric field distributionmap, showing a top view of the electrodes of FIG. 10 in an energizedstate.

FIGS. 11B-D are schematic diagrams demonstrating falloff of the electricfield distribution in the third dimension, showing an exemplary electricfield distribution in the xz-plane (FIG. 11B), in the xy-plane at themidpoint of the electrodes, and in the xy-plane at the tips of theelectrodes.

FIG. 12A is a schematic drawing showing a representative geometry of thetreatment area in which compiled ellipsoids (shown in pink) illustratethe electroporation protocol developed to attain the desired treatmentobjectives.

FIG. 12B is a schematic drawing showing a top view of the treatment areageometry shown in FIG. 12A, and further demonstrating the electrodeinsertion paths.

FIGS. 13A-B are respectively schematic diagrams of an electric fielddistribution and a corresponding conductivity map demonstrating ahomogeneous distribution that only changes by 0.1% for visualizationpurposes when irreversible electroporation is accomplished.

FIGS. 14A-B are respectively schematic diagrams of an electric fielddistribution and a corresponding cumulative conductivity mapdemonstrating a treatment region where more than two electrode pairs areenergized and homogeneous distribution only changes by 0.1% forvisualization purposes when irreversible electroporation isaccomplished.

FIGS. 15A-B are respectively schematic diagrams of an electric fielddistribution and a corresponding conductivity map demonstrating aheterogeneous distribution that changes from 0.67 S/m to 0.241 due toelectropermeabilization caused by electroporation.

FIGS. 16A-B are respectively schematic diagrams of an electric fielddistribution and a corresponding conductivity map demonstrating aheterogeneous distribution that changes from 0.67 S/m to 0.241 S/m dueto electropermeabilization.

FIGS. 17A-B are respectively schematic diagrams of an electricconductivity map and corresponding potential thermal damage resultingfrom electroporation at t=0 s.

FIGS. 18A-B are respectively schematic diagrams of an electricconductivity map and corresponding potential thermal damage resultingfrom the electroporation at t=30 s.

FIGS. 19A-B are respectively schematic diagrams of an electricconductivity map and corresponding potential thermal damage resultingfrom the electroporation at t=60 s.

FIGS. 20A-B are two-dimensional (2-D) diagnostic T1 post-contrast MRIscans in which the tumor was traced.

FIGS. 21A-H is a graphic representation of a three-dimensional (3-D)solid representing a tumor volume and displaying the voltageconfigurations that would mainly affect tumor tissue in this particularsituation.

FIG. 22 is a graph showing a Bipolar IRE pulse (100 us duration) withalternating polarity in the middle of the pulse.

FIG. 23A is a schematic diagram of a representative circuit model forswitching polarity between pulses and multipolar pulses.

FIG. 23B is a graph showing the shape of a bipolar pulse that can becreated using the electrical circuit of FIG. 23A.

FIGS. 24A-G are graphs showing various pulsing protocols according tothe invention, demonstrating exemplary frequencies, pulse length, andtime delay between pulses.

FIGS. 25A-B are schematic diagrams showing variations in techniques forgenerating bipolar electrical pulses in accordance with embodiments ofthe invention.

FIG. 25C is a schematic diagram of a representative circuit model forgenerating and administering simultaneous, continuous, but offset pulsesas shown in FIG. 25A.

FIG. 26A is a photograph showing the N-TIRE electrodes with attachedfiber optic probes, which were used in this intracranial treatment ofwhite matter to measure temperature during pulse delivery.

FIG. 26B is a graph showing temperature [° C.] distribution during anN-TIRE treatment in the white matter of a canine subject.

FIGS. 27A-B are graphs showing output of the arbitrary functiongenerator prior to signal amplification by the high voltage MOSFETpositive and negative polarity switches.

FIGS. 28A-B are micrographs showing in vitro experimental results onelectroporation with high-frequency bipolar, pulses using a trypan bluedye exclusion assay.

FIGS. 29A-C are waveforms of IRE with unipolar pulses and high-frequencyIRE with the corresponding TMP development across the plasma membrane(Φ_(pm)) for a 1500 V/cm unipolar pulse (FIG. 29A) and a 1500 V/cmbipolar burst without a delay (FIG. 29B) and with a delay (FIG. 29C).

FIG. 30 is a graph comparing time above the critical threshold (Φ_(cr))for IRE at various center frequencies.

FIGS. 31A-C are waveforms of IRE with unipolar pulses and high-frequencyIRE with the corresponding TMP development across the plasma membrane(Φ_(pm)) for a 1500 V/cm unipolar pulse (FIG. 31A), a 1500 V/cm bipolarburst without a delay and with a shortened negative phase (FIG. 31B),and a 1500 V/cm bipolar burst with a delay and with a shortened, loweramplitude negative phase (FIG. 31C).

FIG. 32 is a chart showing an exemplary output from an in vivo treatmentof the brain with high-frequency, bipolar pulses, where the snapshot istaken within a single burst.

FIGS. 33A-C are schematic diagrams showing electric field, norm (V/cm)contours predicted by the FEM during a 1000 V amplitude burst with acenter frequency of 1 kHz (FIG. 33A) and 1 MHz (FIG. 33B). In FIG. 33C,the homogeneous solution is shown for a constant pulse.

FIGS. 34A-C are magnetic resonance imaging (MRI) images of tissue afternon-thermal IRE on canine tissue. The images show that non-thermal IREdecellularization zones were sharply demarcated T1 iso- to hypo-intense,T2 hyperintense and mild and peripherally contrast enhancing followingintravenous administration of gadolinium, consistent with fluidaccumulation within decellularization sites and a focal disruption ofthe blood-brain-barrier. FIG. 34A shows an MRI before IRE, T2 weighted;FIG. 34B shows superficial non-thermal IRE decellularization site, T2weighted; and FIG. 34C shows post-contrast T1 weighted; the dog's rightis conventionally projected on the left.

FIG. 35 is shows an ultrasound image of brain tissue 24 hour post-IREtreatment. The IRE decelluarization zone is clearly visible as a welldemarcated, hypoechoic circular lesion with a hyperechoic rim.

FIG. 36 is a photograph of fixed brain sections to show position andcharacter of decellularized volume.

FIGS. 37A-B depict images of brain tissue after non-thermal IREtreatment. FIG. 37A shows a sharp delineation of brain tissue showingthe regions of normal and necrotic canine brain tissue after IRE. FIG.37B shows IRE treated brain tissue showing sparing of major bloodvessels.

FIG. 38 shows a three-dimensional MRI source reconstruction of asuperficial lesion site.

FIGS. 39A-E depict various exemplary embodiments of a device accordingto the invention. FIG. 39A depicts a device, showing a connector,wiring, and electrodes disposed at the tip. FIGS. 39B-E depictalternative placement of electrodes, which can be retractable.

FIGS. 40A-C depict an expanded view of an electrode tip according to oneembodiment of the invention. FIG. 40A depicts an exploded view of thevarious concentric layers of materials making up the electrode tip. FIG.40B depicts a side view of the electrode of FIG. 40A, showing thevarious layers in cut-away fashion. FIG. 40C depicts the electrode tipviewed along the proximal-distal plane.

FIGS. 41A-B depict an embodiment of an assembled electrode tip for anexemplary treatment where the tip is inserted within a tumor embeddedwithin benign tissue. FIGS. 41A and 41B depict an embodiment of thedevice of the invention, comprising a hollow core for delivery ofbioactive agents.

FIG. 42 depicts yet another embodiment of a device according to theinvention, in which the outer, non-conductive sheath is adjustable toallow for selection of an appropriate depth/length of electricallyconductive material to be exposed to tissue to be treated. Theembodiment includes screw tappings (not shown) to allow real-timeadjustment of the electrode layer lengths to customize electrodedimensions prior to a procedure.

FIG. 43 depicts an exemplary system according to the invention, whichincludes an adjustable height electrode, a handle for physician guidanceof the device into the patient, and a power source/controller to provideand control electrical pulses.

FIGS. 44A-J depict electrical field outputs for various combinations ofelectrodes emitting different charges. FIG. 44A depicts atwo-dimensional display for the use of four electrodes of alternatingpolarity. FIG. 44B depicts an axis symmetric display for the use of foursimilar electrodes of alternating polarity. FIG. 44C depicts atwo-dimensional display for the use of four charged electrodes, thecenter two at 5000V and 0V and the outer two at 2500V. FIG. 44D depictsan axis symmetric display for the use of a similar electrode set up asFIG. 44C. FIG. 44E depicts a two-dimensional display for the use ofthree electrodes with the center one at 2500V and the outer two at 0V.FIG. 44F depicts an axis symmetric display for the use of threeelectrodes similar to FIG. 44E. FIG. 44G depicts a two-dimensionaldisplay for the use of three charged electrodes, the center at 0V, theleft at 5000V, and the right at 2500V. FIG. 44H depicts an axissymmetric display for the use of a similar electrode set up as FIG. 44G.FIG. 44I depicts a two-dimensional display for the use of three chargedelectrodes, the center at 1750V, the left at 3000V, and the right at 0V.FIG. 44J depicts an axis symmetric display for the use of a similarelectrode set up as FIG. 44I.

FIGS. 45A-C depict thermal effects from use of two bipolar electrodesand an intervening balloon.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

Irreversible electroporation (IRE) is a new focal tissue ablationtechnique. The treatments are capable of sparing major blood vessels,extracellular matrix and other sensitive or critical structures. Theprocedure involves the delivery of low-energy electric pulses throughminimally invasive electrodes inserted within the tissue. The targettissue is exposed to external electric field distributions around theelectrodes, which alter the resting transmembrane potential of thecells. The degree of tissue electroporation (i.e., no effect, reversibleelectroporation and/or irreversible electroporation) depends on themagnitude of the induced transmembrane potential.

Numerical models for electric field optimization are available andtypically include the physical properties of the tissue and treatmentparameters including electrode geometry and pulse parameters (e.g.,duration, number, amplitude, polarity, and repetition rate). Thesemodels can also incorporate the dynamic changes in tissue electricconductivity due to electroporation and thermal effects.

In embodiments of the invention there is provided a numerical model tovisualize the IRE treated regions using sequential independentcombinations of multiple energized and grounded electrodes.Specifically, in such models electric conductivity changes due toelectroporation and thermal effects from an IRE pulse sequence arecapable of being incorporated into the analysis for developing andconstructing more effective treatment protocols. A particular embodimentinvolves setting the resulting conductivity distribution as the initialcondition for the next pulse sequence, then repeating this proceduresequentially until all the pulse sequences are completed. In thismanner, electric conductivity dependencies from previous pulses areincorporated and more accurate electric field distributions arepresented. It is important to note that it is assumed that once a tissueis irreversibly electroporated, the tissue conductivity would not revertback. Consequently, a comprehensive IRE distribution can be presented inwhich the conductivity changes due to the previous pulses areconsidered. Such methods are most useful when using three or moreelectrodes with electrode-pairs being energized independently.

The electric conductivity map in certain circumstances can be crucial inthe treatment planning of irreversible electroporation and other pulsedelectric field therapeutic applications. The conductivity map is whatdetermines how the current generated by the applied voltages/potentialswill flow and the magnitude of the electric field. Several factorsaffect this distribution before, during and after the treatmentincluding tissue heterogeneities, electropermeabilization, thermaleffects and multiple treatments.

First, each tissue has its own “resting/unique” electric conductivitybefore the application of the electric pulses. Thus, in any particularorgan or system there could be a mixture of conductivities that need tobe accounted for in the treatment planning as in the case of whitematter, gray matter and tumor tissue in the brain for example. Also, dueto the permeabilization of the cells in the tissue that are exposed toan electric field threshold capable of altering the membrane structure,there is an increase in conductivity as well (electroporation effect).In addition, each of the tissue's conductivity will vary with changes intemperature as is the case for brain (3.2% C⁻¹) or liver (2% C⁻¹).

The main region treated by irreversible electroporation does not havesufficient increase in temperature to generate thermal damage, however,at the electrode tissue interface (where the electric field is highest)there is a significant increase in temperature and thus the conductivitymap is altered. Capturing these and other dynamic effects can be crucialsince they represent more accurate/realistic treatment geometries andpulse parameters that are not captured elsewhere. Accounting for theseeffects in treatment planning software is expected to lead to theoptimization of pulse parameters and minimize damage to surroundinghealthy tissue.

Numerical modeling methods, such as finite element modeling (FEM), aremore accurate and are actually where the previous treatment planningsystems derive their solutions (the lookup table and analyticalsolutions are calibrated to mimic the numerical solutions).

The reason numerical solutions were not implemented previously is thatsoftware packages to do so can be expensive, can take extensive periodsto come up with a solution (inhibiting real-time feedback as was thegoal above), and require familiarity with complex software in order todevelop protocols (practically requiring an engineer to develop theplans). Because the direction of EBTs is toward application in morecomplex settings where more accurate solutions are desirable and takepriority over time for development, the system described in thisdisclosure is one that can be performed with numerical solutions bydeveloping the treatment plan well in advance (hours, days, weeks, ormonths) of its implementation.

Example I

General Stages of Planning Electroporation-Based Treatments.

A canine patient with a 360 cm³ tumor in the left thigh was treatedaccording to a treatment planning embodiment of the invention. Thistreatment plan serves to demonstrate the complexity and numerous stepstypically involved in developing and implementing a comprehensivetreatment plan for electroporation-based therapies. This description isintended to provide guidance as to the formulation of a basic treatmentplanning system, which can be operably configured to include one or moreof the following stages:

Image Acquisition. Images of the target lesion or of a portion of thebody to be treated can be acquired by taking an array of medical imagesusing one or more imaging modalities, including CT, MRI, PET, or US toname a few.

As shown in FIG. 3 , in preparation for isolating and reconstructing atarget region, an imaging modality such as computed tomography CT can beused to determine the presence of a tumor. Using an image or series ofimages, information about or relating to the region of interest can becollected and used to determine a targeted region, its location, itsposition, any important or relevant nearby structures that must beaccounted for (such as blood vessels, nerves, collecting ducts, etc.),and any relative basic dimensions (such as depth within tissue, basiccross-sectional sizes, distance from other structures, etc.). The CTimages shown in FIG. 3 have used axial slices with TeraRecon software tocompile the pixels into voxels and develop other sectional slices aswell as an overall 3D reconstruction of the scans based on radiodensity(though individual regions of interest have not been isolated). Otherimaging modalities such as ultrasound or MRI may also be used to assessthe lesion.

Regions of Interest (ROI) Tracing. The target ROI can be outlined in theimages used to identify the tumor, whether manually or by way of acomputer program, to identify a potential treatment area. For example, acomputer program capable of detecting anomalies, such as the OsiriXopen-source image analysis software (Geneva, Switzerland), could be usedto outline the targeted region (e.g., a tumor, site forelectrogenetransfer, etc.). As shown in FIG. 4 , one of the CT scansfrom FIG. 3 is shown with the region of interest traced. Tracing theregion of interest in each of a series of CT images compiling the 2Dtraces of each slice would allow for compilation of 3D geometry for thetarget region.

Visualizing and Reconstructing 3D Geometry. The traced regions ofinterest from a series of axial CT slices can be compiled andinterpolated between the steps to create a three-dimensional geometrythat the practitioner could use to gain an understanding of the basicshape of the target mass and/or its location relative to other tissues.

FIG. 5 shows a series of axial traces having been compiled to create arepresentative shape of the targeted region in three dimensions. Thisreconstruction may be maneuvered to assess its general shape and thusallow determination of potentially efficient electrode insertionapproaches.

If desired, the reconstructed geometry can also be visualized relativeto the rest of the patient. This allows one to assess (in greater detailthan the initial FIG. 3 images) physical constraints such as bonespreventing electrode insertion, relative location of sensitivestructures, and orientation of the lesion relative to the body, allowinga practitioner to evaluate optimal electrode insertion approaches. Forexample, in FIG. 6 , the long axis of the tumor is roughly parallel tothe length leg and femur, so a user may consider reducing the number ofelectrodes and insertions used by orienting the electrodes along thisaxis, or they may go with more electrodes perpendicular to the top ofthe leg (since the femur prevents access from the bottom of the leg).

Geometry Modeling. The 3D geometry can then be imported into finiteelement modeling software (FEM). Indeed, several geometries can beimported using software such as Comsol Multiphysics (Comsol, Stockholm,Sweden), including: a) just the targeted region or mass; b) the targetedregion and other traced neighboring regions (muscle, fat, bone, etc); ora 3D Map of all the voxels to be treated as independent elements in thefinite modeling software. The coordinate system from the medical imagescan also be matched.

FIG. 7 shows a model of the 3D target geometry as imported intonumerical modeling software. More particularly, the geometry developedand shown in FIG. 5 may be converted to a surface or a solid andimported into numerical modeling software. Here, the black shape is aconverted geometry within Comsol Multiphysics for the targeted regionreconstructed above. Its dimensions and volume have been normalized toensure its size matches that of the reconstructed volume.

Assign Model Properties. Any physical and/or thermal properties and/orelectrical properties can be assigned in numerous ways. For example, theproperties can be assigned arbitrarily; deduced by designating which ofthe target region or the other traced neighboring regions are of whattissue type and using properties of these tissue types from theliterature; experimentally measured with a “pre-pulse” (e.g., asdescribed in U.S. patent application Ser. No. 12/491,151, “IrreversibleElectroporation to Treat Aberrant Cell Masses;” or the properties can bederived from an algorithm or coordination scheme based on voxel or pixelvalue imported from the 3D map.

The assignment of properties to the model can be performed withinsoftware and manually accounted for in placements. If such propertiesare either assigned arbitrarily or are deduced as described above, thedifferent shapes depicted in the model (e.g., FIG. 7 ) may each beassigned a different set of properties to best represent the tissue ormaterial used (such as 0.025 S/m for the fatty tumor, and 0.5 S/m forthe surrounding tissue).

In a preferred embodiment, the tissue properties are derived frommedical images. Due to the properties of tissue and how the tissues areassessed by modern imaging techniques, it may be possible to deriveaccurate estimations of a tissue's properties based on its response tothe various imaging modalities.

For example, for computed tomography, pixel values are based on theradiodensity of the tissue at that point in the image (its attenuation).It is common practice to scale these attenuations relative to distilledwater according to the equation:

${HU} = {\frac{\mu_{X} - \mu_{water}}{\mu_{water} - \mu_{air}} \times 1000}$

where μ_(X), μ_(water), and μ_(air) are the linear attenuationcoefficients of that point in the tissue, water, and air, respectively.Essentially, this system normalizes the radiodensity of all tissuesrelative to water.

A tissue's Hounds Unit (HU) value may serve as a representation of itsrelative water content, with larger absolute value HU's (because it canbe negative as well) containing less water. Thus, one could determine(with some noise) a function of HU that goes in the domain from −1000(air, minimal radioattenuation) to +1000 (an equivalent difference ofhigher radioattenuation), where the curve estimates the water content.The data in Table I supports this concept.

TABLE 1 Substance HU Conductivity, S/m (from literature) Air −1000 0 Fat−120 0.025 Water 0 — Muscle +40 0.5 Bone >+400 .0025

These are general evaluations of conductivity. It does not matter whatthe conductivity of distilled water is, but it would likely be taken tobe that of physiological saline for conductivity estimation (1.2 S/m).From qualitatively assessing the data in Table I, it can be seen thatthe closer a tissue's HU is to 0, the higher its conductivity. This isalso reflected because it is known that muscle has a higher watercontent than fat, which is closer to the HU of 0 and a higherconductivity, while bone having the least water content of all, is theleast conductive.

Although not a comprehensive table, the trends are clearly evident thatone may be able to fit an interpolation function between HU andconductivity. With further exploration, it may be seen that this may bea result of higher volumetric water concentrations having higherconductivity. The idea that higher percentage of water causes a tissueto have a radiodensity more similar to that of water is an assumption,but when taking it into account, the relationship between conductivityand %-water may also be used to estimate the tissue's electricalproperties, as described in Duck, 1990 (FIG. 8 ).

Evaluate any Physical Placement Constraints. Potential physicalplacement constraints, such as vital structures (nerves, brain, bloodvessels, etc.), access orientation preferences (from head, from rear,supine, prone patient positioning, etc.), and/or physical barriers(bones, sensitive structures, etc.) can be identified. The potentialconstraints can then be used to guide/constrain what angles are possiblefor the electrodes and if the electrodes should be placed to avoidcertain areas more than others.

FIG. 9 shows a graphic 3D reconstruction of the target tumor in relationto surrounding structures within the body, which is useful fordeveloping treatment constraints. The physical location of the tumorrelative to the rest of the body (shown in FIG. 9 by arrows pointing outvasculature and nerves, for example) can be demonstrated using thepreviously prepared 3D geometric representation of the tumor. Thisinformation may be used to constrain or direct where the electrodesshould be placed and give priority to regions that should be sparedrelative to regions that would not cause as significant of problems.

Placement of Electrodes. Any number of electrodes could be placed intoor around the targeted region. Their number, location, orientation, andsize could all be adjusted independently.

FIG. 10 is a graphic 3D representation of the imported tumor geometrywith electrodes placed. Here, the geometric representation of thetargeted region is depicted in red, while representations of electrodesare shown at two locations in blue. The number, orientation, andlocation of these electrodes is capable of being manipulated to satisfythe desired treatment objectives.

Simulation of the Electric Field Distribution. Simulation of theelectric field distribution (e.g., numerically solved electric fielddistributions) are capable of being correlated with experimental data tosuperimpose predicted volumes of affected regions (treated, untreated,thermal damage).

For example, FIG. 11 shows the electrodes depicted in FIG. 10 in anenergized state. For the electrode on the left, a section on the end hasbeen set to a voltage while a section on the rest has been set to groundwith a section of insulation between, creating a voltage gradient thatsurrounds the single needle. For the pair of electrodes on the right,the entire length of one electrode has been set to a voltage while theother electrode has been set to ground, creating a voltage gradientbetween them.

The color maps are representative electric field isocontour regions thatmay be used in determining predicted treatment regions, reversibleregions, or safety margins based on electric field thresholds. Forexample, if the protocol anticipates an IRE electric field threshold of500 V/cm, then the entire volume of the tissue exposed to this electricfield or higher (depicted in green) would be the predicted treatmentregion. In addition, if it were desired to ensure sparing of a sensitivestructure such as a nerve, and an exact resolution of theabove-predicted 500 V/cm IRE electric threshold was insufficient toguarantee sparing, a different electric field may be used to predict asafety margin which would be used to ensure that this threshold is notcrossed by the sensitive structure (such as 250 V/cm depicted in red).

Another factor to consider in any analysis for determining properplacement of the electrodes and/or the charge needed for a particularapplication is the expected behavior of the electric field relative tothe electrodes. As shown in FIGS. 11B-D, the electric field distributionis typically at a maximum at the cross-sectional region midway betweenthe lengths of the electrodes and tapers off toward the ends of theelectrodes.

More particularly, the image shown in FIG. 11B shows the electric fielddistribution between 35000 and 150000 V/m looking at both electrodessimultaneously in the xz-plane. The grey rectangles are the electrodes,running along the z-axis, and separated by 1.5 cm (center-to-center)along the x-axis. Here, one can see that the electric field is greatestat z=0, and decreases as one moves towards the tips of the electrodes.The 2-dimensional cross-sectional estimates are calibrated to representthe electric field distribution at z=0, and do not take into account thelosses that occur over the length of the electrode.

FIGS. 11C and D in comparison show x-y cross-sectional plane view of theelectric field distribution at z=0 and the x-y cross-sectional planeview electric field distribution at z=1 cm (the tips of the electrodes),respectively. By comparing these two distributions, it can be seen thatthe electric field distribution decreases as distance from the center ofthe electrode lengths increases. To accurately predict the treatmentregions in three dimensions, these differences should be taken intoaccount for the overall 3D nature of treatments. The methods, systems,and devices according to the invention include consideration of thisfactor.

Evaluate Success of Outcome. Determine whether the setup usedappropriately meets its treatment demands of affecting the desiredregions while preventing unacceptable effects on untargeted andsensitive regions. This could be assessed qualitatively orquantitatively with a fitness function.

Optimization. The evaluation of physical constraints, placement ofelectrodes, simulation of the electric field distribution, andevaluation of outcome success can be repeated until a suitable solutionis developed. This optimization stage can be performed manually(interactively) by a practitioner or automatically. The OptimizationQuality Function of Formula I discussed in more detail below could alsobe used for manual optimization.

In this embodiment, the optimization phase of the system was performedqualitatively and was iterated with the previous four steps untilsettling on the electrode array shown in FIGS. 12A and B. In thisembodiment, the resultant representative geometry of compiled ellipsoids(shown in pink) illustrates the satisfactory electroporation protocoldeveloped in order to attain the desired treatment objectives. In FIG.12A, it can be seen that a highly complex array of electrodes (blue) wasselected, where some electrodes are inserted and exposed an amount (suchas 1 or 2 cm), to treat an amount of depth with pulsing, beforewithdrawing them some and repeating the pulsing. This was done to ensurecomplete treatment along the depth of the treatment. The blue cylindersdepict discrete electrode placements for pulsing, and the ones stackedon top of each other represent this aspect.

Further, as shown in FIG. 12B, a top view of the graphic representationof the treatment area of FIG. 12A is provided, in which the electrodeinsertion paths can be seen. Since the electrodes were all runningperpendicular, spacing dimensions have been outlined to aid theplacement of the electrodes for the practitioner. The pulses would beadministered between each electrode and the electrodes in closestproximity to it. Electric pulse parameters are adjusted between eachelectrode firing pair based on separation distance and the desiredtreatment region (based on targeted volume and avoidance of sensitivetissues). The dimensions in red are also used as guidelines for theplacement of the outer electrodes relative to the margins of the tumorto prevent excessive treatment of peripheral (untargeted) regions.

Implementation. Once a desired solution has been developed, thegenerator system for applying the designated pulsing protocol can be setup for implementation of the desired protocol. More particularly, thepractitioner could then place the electrodes according to the prescribedprotocol and let the generator apply the pulses.

Also during implementation, the systems, methods, and or devicesaccording to the invention can be operably configured to monitor certainvariables. One such variable can include monitoring the temperature ofthe electrodes and/or surrounding tissue in real time during treatmentto ensure limited to no thermal damage to the tissue being treated. Ifmonitored in real time, adjustments could then be made, if necessary, toavoid damage.

One, multiple, or all phases of system embodiments according to theinvention can be performed manually or be performed (in whole or in anynumber of parts) by an automated system capable of performing the phasesfor the practitioner. Many of these steps can be performed without userinput, and could be blocked off into distinct automated processes(with/without coupling to human-performed processes) or could be linkedtogether through a comprehensive system. All of this is able to be donefor an initial treatment, or redone for any retreatments that may benecessary, with or without new images (depending on case circumstances).

Example II

Comprehensive Package System: Treatment Planning Software.

Due to the great complexity and time required to develop customizedtreatment protocols for each patient, it is desirable to automate one ormore steps, or the entirety, of the treatment planning process. SinceCassini Oval and other analytical solutions are limited by their abilityto incorporate many of the complexities commonly found in treatmentsituations (such as heterogeneities, complex geometries, differentelectrode dimensions and orientations, etc.), and because the trendseems to be to move treatment planning towards a simpler solution forpractitioners so less time is wasted in trying all the differentavailable options—a robust automatic treatment planning system thatincorporates numerous variables and runs a self-optimization algorithmto automatically determine the optimal treatment parameters needed to beused to treat a particular patient is highly desired.

Systems according to embodiments of the invention are flexible in thatsuch systems can be operably configured to solve many scenariosnumerically and to select the best electrode geometry and pulseparameters for a given situation. Alternatively or additionally,solutions may be obtained analytically, with tables, etc.

Embodiments of the systems according to the invention can be operablyconfigured to be run on an independent system well in advance oftreatment administration to allow sufficient computation time, review,and possible re-working of the protocol prior to treatment. Theappropriate protocol could then be uploaded directly to the pulsegenerator.

Model Creation. Preferred embodiments of systems according to theinvention include a model creation stage for establishing an initialmodel of the target area.

Geometry: Treatment geometries (information, such as tumor dimensions,electrodes, and peripheral tissue dimensions, for example) may be inputmanually, by analyzing medical images that were taken and anyreconstructions, from computer analyses of medical images/tomography, orother (2D and 3D) mapping techniques.

Properties: Conductivity values for the model subdomains may be obtainedby measuring them on the subject directly (placing electrodes withintissue then applying a voltage and measuring the current to get Z/σ), bytaking typical values found in the literature for the tissue types, orby noninvasive a measuring techniques such as functional MagneticResonance Imaging (fMRI), Electrical Impedance Tomography, etc; andcombining these with the relevant equations (for E-field distributions,it is the ratio between tissues/regions that alters the field, absolutevalues will only be important when considering thermal effects). Medicalimages that already obtain the conductivity values (fMRI) or coupled toconductivity values (analyzing and mapping a medical image for thedifferent tissues and coupling the regions to a conductivity valuedetermined as described above) may then be used as the geometries for anumerical/analytical model as the various subdomains, to establish theinitial model.

Electrodes. Once the model geometry has been developed, a single or anyset of electrode options (type, number, dimensions, etc.) may beselected to be used or allowed to be selected by the program.

Running the Program. After setting up the geometry and electrode optionsto consider, the practitioner would essentially select a “GO” button tolet the program run through the many variations to use and solve eachusing FEM or advanced analytical methods. The program would solve eachscenario for various effects (no effect, reversible electroporation,irreversible electroporation, or thermal) and distributions within themodel. Thermal considerations will greatly increase the computationalcost of the model, but may be desired to determine thermal damage andscarring, especially in very sensitive structures.

Exemplary Optimization Quality Function. The systems of embodiments ofthe invention can employ a variety of algorithms (iterative, genetic,etc.) in order to optimize the treatment parameters for the bestpossible result for a particular patient scenario. Such systems can alsobe operably configured to employ a function for evaluating the qualityof each solution, where desired results, D, (IRE and/or REB throughoutthe targeted regions) are added; and the undesired results, U, (thermaldamage, IRE beyond targeted region, etc.) are subtracted, with eachaspect having its own unique scaling (since IRE to entire targetedregion is far more important that avoiding IRE to healthy tissues). Onesuch function can include:

ψ(ET,EP,ϕ, . . . )=A·[IRE] _(D) +B·[REB] _(D) −C·[Therm]_(D)−E·[IRE]U−F·[REB] _(U) −G·[Therm]U  Formula I:

wherein D=Desired/Targeted Volume (done as a percentage);

U=Undesired/Peripheral Volume (done as an absolute value);

A, B, C, E, F, G=Scaling factors, with likely situations including: 1) A& B>>C, E, F, and G (treatment success most important); 2) G>>C (thermalto healthy worse than to targeted; 3) B & F may be neglected in purelyIRE treatments; and 4) F can typically be assumed to be=0 since nonegative effects to the tissue would be associated with this parameter,since it would either have no effect (without chemicals), or will nothave an effect on healthy cells (with selective chemicals); but maymatter in situations involving nonselective chemicals;

ET=Electrode Type and geometry (single/dual, diameter, length);

EP=Electrode Positioning (location and orientation in 3D space);

ϕ=Applied voltage;

ψ=Quality, the value of the protocol on the entire domain of thetargeted and surrounding volumes.

Additional details on ψ(ET, EP, ϕ, . . . ): This is the value functionof a given treatment protocol for the modeled domain previouslymentioned as a function of electrode type and geometry, electrodepositioning, applied voltage, and any other factors. Morespecifically: 1) ET(style, number, dimensions), with style referring tothe style of the pulse, such as single, multi-unipolar, hybrid,proprietary, etc., with number referring to the number of probes used,and dimensions referring to the geometry and dimensions of all exposedand insulated regions in all three directions for each electrode used.

EP refers to the position of each or all electrodes in relation to areference point arbitrarily chosen within the (x, y, z) domain of themodel (location and orientation). In one example, the center of thetumor could be selected as the reference point and arbitrarily set to(0, 0, 0). The reference point may also be selected ahead of time orafterwards by the practitioner that will be easy for the practitioner tophysically use at the time of treatment administration, such as someanatomical landmark that can be used as a reference for where theelectrodes are and the electrode orientation. It is also possible tomatch the coordinate system from the medical images.

The ψ function may be solved for altered ET and EP, and the ϕ may thenbe scaled accordingly for the geometry (since it the model geometry andproperties that will affect the shape of the distribution, the absolutevalue of it may be scaled to the applied voltage after this shape isfound for each ET and EP). This would dramatically reduce the number ofiterations and thus the computational cost.

In embodiments, the system can be operably configured to iterativelyadjust ET, EP, etc. and obtain the resulting ψ, storing the top ones (orall those meeting some type of baseline threshold criterion). Theresulting stored solutions would then be saved for presentation to thepractitioner for conducting a review and visually assessing the value ofeach solution for selecting the protocol that best meets the demands ofthe therapy (could range on their arbitrary criterion such as the bestquality, most simple to administer and apply the EP in the treatment,most robust, etc.)

The electrical parameters used (number of pulses, repetition rate,shape, pulse length, etc.) can be set as standardized parameters fortypical treatments, and optionally these parameters can be flexible incase certain scenarios require different values—such asabdomino-thoracic procedures requiring repetition rate to besynchronized with the patient's heart rate to reduce the risk ofpulse-induced arrhythmias. If known or found experimentally, standardelectrical parameters can be used to determine the best combination oftreatment parameters to use and have been applied to varioustissues/tumors to determine the electric field threshold of each forthis set of parameters, thus allowing treatment outcome to be reviewedand not just electric field distributions. Table II provides a list ofexemplary electric parameters that can be manipulated within the IREtreatments discussed herein.

TABLE II Parameters Pulse length: ns-ms range Number of pulses: 1-50,000pulses Electric Field Distribution: 1-5,000 V/cm Frequency of Pulse0.001-1000 Hz Application: Frequency of pulse signal: 0-100 MHz Pulseshape: square, triangular, trapezoidal, exponential Pulse type: decay,sawtooth, sinusoidal, alternating Electrode type: polarity Positive,negative, neutral electrode charge pulses (changing polarity withinpulse) Multiple sets of pulse parameters for a single treatment(changing any of the above parameters within the same treatment tospecialize outcome) Parallel plate: 0.1 mm-70 cm diameter (and largerfor applications relating to e.g., whole organ decellularization) Needleelectrode(s): 0.001 mm-1 cm diameter Single probe with embedded diskelectrodes: 0.001 mm-1 cm diameter Spherical electrodes: 0.0001 mm-1 cmdiameter Needle diameter: 0.001 mm-1 cm Electrode length (needle): 0.1mm to 30 cm Electrode separation: 0.1 mm to 5 cm, or even 5 cm to 20 cm,or 20 cm to 100 cm, and larger (for reversible electroporation, genedelivery, or positive electrode with ground patch on patient's exterior,e.g.)

Additional considerations, such as multiple pulse protocols that createdynamic tissue properties as a function of electric field, andtemperature changes, may need to be investigated or added. Such dynamicproperties are demonstrated in FIGS. 13-19 .

FIGS. 13A-B demonstrate a situation in which there would be little to nochange in the physical properties of the tissue as a result ofelectroporation. More specifically, as shown in FIG. 13A, an electricfield distribution [V/cm] generated by an applied voltage difference of3000V over the upper two electrodes is shown. FIG. 13B provides aconductivity map [S/m] displaying a homogeneous distribution that onlychanges by 0.1% for visualization purposes when irreversibleelectroporation is accomplished. The white outline represents the regionof tissue that is exposed to an electric field magnitude that issufficient for generating irreversible electroporation.

FIGS. 14A-B demonstrate an electric field distribution and conductivitymap for a treatment region for a given situation in which more than twoelectrode pairs are energized. In FIG. 14A, an electric fielddistribution [V/cm] generated by an applied voltage difference of 3000Vover the right two electrodes is shown. FIG. 14B shows a conductivitymap [S/m] displaying a homogeneous distribution that only changes by0.1% for visualization purposes when irreversible electroporation isaccomplished in this set up. In FIG. 14B, a cumulative visualization ofthe treatment region is shown.

FIGS. 15A-B demonstrate a change in the shape and size of the treatmentregion due to electropermeabilization. More particularly, in FIG. 15A,an electric field distribution [V/cm] generated by an applied voltagedifference of 3000V over the upper two electrodes is shown. FIG. 15Bprovides a conductivity map [S/m] displaying a heterogeneousdistribution that changes from 0.67 S/m to 0.241 due toelectropermeabilization as a result of electroporation. Of particularnote in this example, the shape and size of the treatment region isconsequently adjusted as a result of this change. In FIG. 15B the shapeand size of the planned treatment region is different than in the aboveexamples (FIGS. 13B and 14B) in which the conductivity was assumed toremain constant throughout the delivery of the pulses.

FIG. 16A provides an electric field distribution [V/cm] generated by anapplied voltage difference of 3000V over the right two electrodes, whileFIG. 16B shows a conductivity map [S/m] displaying a heterogeneousdistribution that changes from 0.67 S/m to 0.241 S/m due toelectropermeabilization. Of particular interest, the first set of pulsesusing the top two electrodes increased the conductivity of the tissuewhich in turn modified the electric field distribution (i.e., treatmentregion) for the second application of pulses (right two electrodes)adjacent to the permeabilized region.

The following examples are different than the previously describedexamples in which the treatment region depended onelectropermeabilization, and multiple electrode combinations. In thiscase, a 2-D model of an irreversible electroporation protocol is shownin which the electric parameters of the protocol included 90 pulses, at2000V, delivered at a frequency of 1.5 Hz, using 100 μs pulses. The 2Dmodel generates much higher temperatures and thus changes relative tothe complete 3D model since the heat has a larger volume in which todiffuse. Nevertheless, this case is reported for illustration purposesand to show that in fact these dynamic effects can be incorporated intotreatment planning models. Changes only due to temperature areincorporated in this example to emphasize the importance of accountingfor these effects in the models.

To illustrate the thermal effect of electroporation on tissuescontacting the electrodes, FIGS. 17-19 are provided. FIG. 17A providesan electric conductivity [S/m] map at t=0 s in which the irreversibleelectroporation area is 2.02 cm². FIG. 17B shows a thermal damageassessment by the potential increase in temperature due to the electricpulses which occurs when greater than 0.53.

FIG. 18A provides an electric conductivity [S/m] map at t=30 s in whichthe irreversible electroporation region is 2.43 cm². FIG. 18B shows somethermal damage visualized at the electrode-tissue interface 0.11 cm².

FIG. 19A provides an electric conductivity [S/m] map at t=60 s in whichthe irreversible electroporation area is 2.63 cm². FIG. 19B showssignificant thermal damage at the electrode-tissue interface due tothermal effects 0.43 cm².

Therapy Application. Once the practitioner has selected a desiredsolution from the options on the treatment planning software, theelectrical protocol (pulse characteristics, number, sequence, etc.)could be saved and then uploaded to the pulse generator system. At thispoint, the practitioner would have to do no more than place theelectrodes in the predetermined positions and hit “START”, at whichpoint the instrument carries out the prescribed pulsing conditions.

Example III

Exemplary Methods for IRE Treatment Planning

Open source image analysis software (OsiriX, Geneva, Switzerland) wasused to isolate the brain tumor geometry from the normal brain tissue.The tumor was traced in each of the two-dimensional (2-D) diagnostic T1post-contrast MRI scans as shown in FIGS. 20A-B. Attempts were made toexclude regions of peritumoral edema from the tumor volume by compositemodeling of the tumor geometry using all available MRI sequences (T1pre- and post-contrast, T2, and FLAIR) and image planes.

As provided in FIGS. 21A-H, a three-dimensional (3-D) solidrepresentation of the tumor volume was generated using previouslyreported reconstruction procedures. The tumor geometry was then importedinto a numerical modeling software (Comsol Multiphysics, v.3.5a,Stockholm, Sweden) in order to simulate the physical effects of theelectric pulses in the tumor and surrounding healthy brain tissue. Theelectric field distribution was determined in which the tissueconductivity incorporates the dynamic changes that occur duringelectroporation. In this model, a 50% increase in conductivity wasassumed when the tissue was exposed to an electric field magnitudegreater than 500 V/cm, which has been shown as an IRE threshold forbrain tissue using specific experimental conditions. Currently, thethreshold for brain tumor tissue is unknown so the same magnitude asnormal tissue was used for treatment planning purposes.

Based on the tumor dimensions and numerical simulations, the voltageconfigurations that would mainly affect tumor tissue were determined andare provided in Table III as well as are displayed in FIGS. 21A-H.

TABLE III ELECTRODE NUMBER VOLTAGE ELECTRODE EXPOSURE VOLT-TO-DIST PULSEOF (V) GAP (CM) (CM) RATIO (V/CM) DURATION PULSES FREQUENCY 500 0.5 0.51000 50 μs 2 × 20 ECG synchronized 625 0.5 0.5 1250 50 μs 4 × 20 ECGsynchronized

IRE Therapy. Total intravenous general anesthesia was induced andmaintained with propofol and fentanyl constant rate infusions. A routineleft rostrotentorial approach to the canine skull was performed and alimited left parietal craniectomy defect was created. The craniectomysize was limited to the minimum area necessary to accommodate placementof the IRE electrode configurations required to treat the tumor, asdetermined from pre-operative treatment plans. Following regionaldurectomy, multiple biopsies of the mass lesion were obtained, whichwere consistent with a high-grade (WHO Grade III) mixed glioma.

After administration of appropriate neuromuscular blockade and based onthe treatment planning, focal ablative IRE lesions were created in thetumor using the NanoKnife® (AngioDynamics, Queensbury, N.Y. USA), andblunt tip electrodes. The NanoKnife® is an electric pulse generator inwhich the desired IRE pulse parameters (voltage, pulse duration, numberof pulses, and pulse frequency) are entered. The NanoKnife® is alsodesigned to monitor the resulting current from the treatment and toautomatically suspend the delivery of the pulses if a current thresholdis exceeded.

The electrodes were inserted into the tumor tissue in preparation forpulse delivery. The blunt tip electrodes were connected by way of a6-foot insulated wire (cable) to the generator. After foot pedalactivation, the pulses were conducted from the generator to the exposedelectrodes.

The two sets of pulse strengths were delivered in perpendiculardirections to ensure uniform coverage of the tumor and were synchronizedwith the electrocardiogram (ECG) signal to prevent ventricularfibrillation or cardiac arrhythmias (Ivy Cardiac Trigger Monitor 3000,Branford, Conn., USA). The sets of pulses were delivered withalternating polarity between the sets to reduce charge build-up on thesurface of the electrodes. In addition, shorter pulse durations thanthose used in previous IRE studies were used in order to reduce thecharge delivered to the tissue and decrease resistive heating during theprocedure. Previous calculations and experimental data from previousintracranial IRE experiments ensured that no thermal damage would begenerated in normal brain. The temperature measured near the electrodesshowed a maximum 0.5° C. increase after four sets of twenty 50-μs pulseswhen using similar parameters to the ones in Table I. In addition, thecharge delivered during the procedure was typical or lower than thatused in humans during electroconvulsive therapy, a treatment fordepression that also uses electric pulses.

Example IV

Treatment Systems, Methods, and Devices Using Bipolar Electric Pulses

It has been found that alternating polarity of adjacent electrodesminimizes charge build up and provides a more uniform treatment zone.More specifically, in IRE treatments there is an energized and groundedelectrode as the pulses are delivered. In embodiments, charge build-upon the surface of the electrodes can be minimized by alternating thepolarity between sets of pulses. It is believed that there are stillelectrode surface effects that can be associated with negative outcomes.

Further, the use of bipolar pulses (net charge of zero) as seen in FIG.22 is a way to further minimize the charge delivered to the tissue. FIG.22 is a graph showing a Bipolar IRE pulse (100 us duration) withalternating polarity in the middle of the pulse in order to minimizecharge delivered to the tissue. In this manner, negative effects can beprevented, reduced, or avoided as part of IRE treatment in the brain,including deleterious electrochemical effects and/or excessive chargedelivered to the tissue as in electroconvulsive therapy.

In one experiment, a superficial focal ablative IRE lesion was createdin the cranial aspect of the temporal lobe (ectosylvian gyrus) using theNanoKnifeB (AngioDynamics, Queensbury, N.Y.) generator, blunt tipbipolar electrode (AngioDynamics, No. 204002XX) by delivering 9 sets often 50 us pulses (voltage-to-distance ratio 2000 V/cm) with alternatingpolarity between the sets to prevent charge build-up on the stainlesssteel electrode surfaces. These parameters were determined from ex-vivoexperiments on canine brain and ensured that the charge delivered duringthe procedure was lower than the charge delivered to the human brainduring electroconvulsive therapy (an FDA approved treatment for majordepression).

Other undesirable consequences of various electroporation protocols havealso been experienced. More specifically, with the application ofelectric potentials, electrical forces may drive ions towards oneelectrode or the other. This may also lead to undesirable behavior suchas electrolysis, separating water into its hydrogen and oxygencomponents, and leading to the formation of bubbles at theelectrode-tissue interface. These effects are further exacerbated formultiple pulse applications. Such effects may cause interference withtreatment by skewing electric field distributions and altering treatmentoutcomes in a relatively unpredictable manner. By altering the polaritybetween the electrodes for each pulse, these effects can besignificantly reduced, enhancing treatment predictability, and thus,outcome. This alternating polarity may be a change in potentialdirection for each pulse, or occur within each pulse itself (switch eachelectrode's polarity for every pulse or go immediately from positive tonegative potential within the pulse at each electrode).

FIG. 23A is a schematic diagram of a representative circuit model forswitching polarity between pulses and multipolar pulses. As shown inFIG. 23A, a basic circuit according to embodiments of the invention maycontain a) a generator supply circuit containing a voltage source andcapacitor bank to accumulate sufficient charge for pulse delivery; b) asimultaneous switching mechanism; c) electrodes for pulse delivery(here, 2 electrodes are shown); and d) a parallel capacitor-resistorequivalent to represent the behavior of biological tissues. FIG. 23Bshows an exemplary bipolar pulse that can be created using the circuitof FIG. 23A.

The circuit can be operably configured to function in the followingrepresentative manner. At Time 0, the switches are in position 0. Thevoltage source would be used to charge an array of capacitors to thedesired electric potential for a given pulse. At Time t₁, the switchesmove to position 1. This causes rapid initiation of capacitor discharge,generating a high-slope ΔV between the electrodes placed in the tissue(the first half of a square wave). This gives electrode 1 a “negative”voltage and electrode 2 a “positive” voltage (based on their relativeelectric potentials). The capacitor(s) continue delivering the electriccharge over time, causing a logarithmic decay of the electric potentialto which the tissue is exposed. At Time t₂, the switches move toposition 2. This changes which electrode is connected to which end ofthe circuit, rapidly reversing the polarity of the electric potential,making electrode 1 “positive” and electrode 2 “negative.” The peak ofthis reversal is the same as the remaining charge on the capacitorsafter the decay between t₁ and t₂. The remaining charge on thecapacitors continues to decay. At Time t₃, the switches return toposition 0. This disconnects the circuits, creating a rapid drop in theelectric potential between the electrodes, returning ΔV to zero.Alternatively, at Time t₃, the switch could return to position 1, thenalternate between positions 1 and 2 for a desired period of time todeliver several bipolar pulses in rapid succession. Such switchingcircuitry would enable delivery of a bipolar pulse train comprisingindividual pulses having a duration ranging from 10 ms to 1 ns, muchfaster than any human could achieve.

It should be mentioned that the electric potential difference isarbitrary, and the polarity of any of the pulses in the above-mentionedexample are for demonstration only, and are not the sole method ofobtaining multipolar pulses. Alternative approaches are possible andthis basic circuitry representation may be adapted to generate anyseries of complex pulses by changing the pattern of switch behavior.

For instance, unipolar pulses may have their polarity reversed everypulse or after any number of pulses by moving the switches from position0 to 1 for pulse delivery, then back to 0 (first pulse); then fromposition 0 to 2 for delivery, then back to 0 (second pulse of oppositepolarity). As shown in FIGS. 24A-D, a unipolar pulse of any polarity canbe reversed after one or more pulses up to any number of desired pulsesfor a particular application. For example, a time delay between theunipolar pulse and the reversed polarity unipolar pulse can be anydesired duration as well, including from 5 times the pulse length (FIG.24A), to 3 times the pulse length (FIG. 24B), to 1 time the pulse length(FIG. 24C), to no delay (or effectively no delay) at the time ofswitching (FIG. 24D).

As shown in FIGS. 24E-G, the pattern of alternating between pulsepolarities can be repeated any number of times to accomplish a desiredresult. For example, the bipolar pulse of FIG. 24D is shown repeated attiming intervals of 3 times the pulse length (FIG. 24E), to 2 times thepulse length (FIG. 24F), to 1 time the pulse length (FIG. 24G). Thedelay between bipolar pulses can also be zero (or effectively zero)and/or the bipolar pulses can be repeated any number of time toestablish a particular desired pulsing protocol or pattern.

The pulses could also be made multipolar by switching from position 0 to1 (first polarity), then to position 2 (reversed polarity), then back toposition 1 (returning to initial polarity), and so on, all within thesame pulse.

Even further, the bipolar pulses can be configured in a manner todeliver a charge to the tissue where the net effect of the pulse issomething other than zero. For example, the magnitude of the positiveportion of the pulse can be different than the magnitude of the negativeportion of the pulse. More specifically, the pulse can be 90% positiveand 10% negative or 90% negative and 10% positive. Indeed, any ratio ofpositive:negative charge can be used, including from 0:100 (mono-polarand positive) to 100:0 (mono-polar and negative). Specifically, 50:50(net charge of zero) is preferred, but 90:10, 80:20, 75:25, 60:40 andthe reverse can be used depending on the desired effect.

Additionally, the time between any switch could be used to alter thelength of any pulse or change the pulse repetition rate. And, if varyingcombinations of different capacitor banks were used in the system, thendepending on which ones were connected, it would be possible to changethe applied voltage to the electrodes between pulses or within a pulse(of any polarity).

The shape and type of pulse can also be varied for particularapplications. In various embodiments, the individual electric pulses canbe unipolar while in other embodiments, the individual electric pulsescan be bipolar. In certain preferred embodiments, a train of unipolarpulses is delivered in one direction, followed by a subsequent pulsetrain of opposite polarity. Depending on the outcome desired, thewaveforms of the electric pulses are triangular, square, sinusoidal,exponential, or trapezoidal. Other geometric shapes are contemplated aswell. In some embodiments, an electrode is connected to a system foremploying electrical impedance tomography (EIT), computed tomography(CT), Magnetic Resonance Imaging (MRI), or ultrasound to image thetissue prior to treatment by applying small alternating currents thatthemselves do not damage the tissue.

A large variety of other parameters can influence the efficiency ofmembrane poration, such as the shape of the electrical pulses, polarity,size of target cells, and thermal conditions during and after thepulses.

Another method for avoiding excessive charge build up in tissues beingtreated by electroporation is to deliver counteracting pulsessimultaneously from one or more pulse generator. In embodiments, thepulses delivered by the generators can overlap in time for some portionof the pulse and be offset from one another.

FIG. 25A illustrates the concept of overlapping the equal but oppositecharges delivered from separate pulse generators. In particular, a firstpulse generator administers a first positive pulse for a desired amountof time. Here, the pulse has a duration in the 10 ns to 10 ms range. Atsome time after the first pulse is generated, a second pulse from asecond pulse generator is administered. In this example, the secondpulse is of the same magnitude as the first pulse yet opposite inpolarity. By overlapping the pulses, or simultaneously applying thepulses, the net effect during the overlap is that the tissue does notexperience a charge. In effect the overlap of the pulses creates a delayand the charge delivered to the tissue is only the portion of each pulsethat is outside of the overlap, i.e., the offset.

FIG. 25B illustrates one example of administering opposing polaritypulses from two pulse generators simultaneously, but offset and with nooverlap. As shown, a first positive electrical pulse is initiated by afirst pulse generator. At a desired time following administration of thefirst pulse, a second pulse equal in magnitude to the first pulse butopposite in charge is initiated using a second pulse generator. It isnoted that in this figure that although a summation of the twoindividual signals offset by a delay (pulse duration) is shown, one ofskill in the art could easily incorporate additional signals in order tomanipulate additional pulse parameters. Further, and as with allembodiments described in this specification, the positive and negativeapplied voltages do not have to be of equal magnitude.

In one such embodiment, electrical pulses are delivered in a series oftwo pulses of alternating polarity (from millisecond to nanosecondrange). Use of alternating polarities reduces or eliminates chargebuildup on the electrode(s). For example, two NanoKnife™ (AngioDynamics,Queensbury, N.Y.) devices can be linked to the same electrode array, andprogrammed to deliver synched or slightly offset pulses to theelectrodes. The first pulse can generate a 2500 V/cm electric field of500 ns duration. This pulse is followed immediately (yet slightlyoffset) by the onset of a second pulse, which generates a −2500 V/cmelectric field for 500 ns. The net effect of the pulses in the tissue isa net charge of zero and an additional benefit is avoiding the need forcomplex circuitry as the need for abrupt switching of the polarity isobviated.

Also during implementation of a desired treatment protocol, the systems,methods, and or devices according to the invention can be operablyconfigured to monitor certain variables, such as temperature of theelectrodes and/or surrounding tissue. If monitored during the procedureand in real time, adjustments to the protocol, including adjustments tothe type, length, number, and duration of the pulses, could then bemade, if necessary, to avoid damage of the tissue being treated.

It is important to note that bipolar pulses are only effective forelectroporation if each pulse within the train is long enough induration to charge the plasma membrane to a permeabilizing level. Ifthis is not the case, the pulses offset each other from fully chargingthe plasma, and supra-poration effects dominate when the pulse amplitudeis increased. Additionally, a delay can be included between pulseswithin the train, or the total number of pulses within the train can becontrolled, to limit the Joule heating in the tissue while stilldelivering a lethal dose of energy. Embodiments of the invention areequally applicable to any electroporation-based therapy (EBT), includingtherapies employing reversible electroporation, such as gene deliverytherapy and electrochemotherapy, to name a few. One of skill in the artis equipped with the skills to modify the protocols described herein toapply to certain uses.

The repetition rate of pulse trains can also be controlled to minimizeinterference with, and allow treatment of vital organs that respond toelectrical signals, such as the heart. The concept of alternatingpolarity of pulses can be extended to the use of multiple electrodes.For example, a combination of three electrodes can be used to deliverthree sequential sets of alternating polarity pulses to a target tissue.More specifically, Electrode A can be used to deliver a 500 ns pulse at1000 V at a starting time (T=0) and a 500 ns pulse at −1000 V at T=1 μs.Electrode B can be used to deliver a 500 ns pulse at 1000 V at T=500 ns,and a 500 ns pulse at −1000 V at T=1.5 μs. Electrode C can be used todeliver a 500 ns pulse at 1000 V at T=1 μs, and a −1000V pulse at T=2.0μs. Of course, this concept can be applied using any numbers ofelectrodes and pulse times to achieve highly directed cell killing.

Example V

Monitoring Temperature During Electroporation Procedures

One of the main advantages of N-TIRE over other focal ablationtechniques is that the pulses do not generate thermal damage due toresistive heating, thus major blood vessels, extracellular matrix andother tissue structures are spared. See B. Al-Sakere, F. Andre, C.Bernat, E. Connault, P. Opolon, R. V. Davalos, B. Rubinsky, and L. M.Mir, “Tumor ablation with irreversible electroporation,” PLoS ONE, vol.2, p. e1135, 2007; and J. F. Edd, L. Horowitz, R. V. Davalos, L. M. Mir,and B. Rubinsky, “In vivo results of a new focal tissue ablationtechnique: irreversible electroporation,” IEEE Trans Biomed Eng, vol.53, pp. 1409-15, July 2006, both of which are incorporated by referenceherein in their entireties. The inventors have found that with real timetemperature data measured at the electrode-tissue interface, thenon-thermal aspect of the technique can be confirmed. One such way tomeasure temperature in-vivo during the pulse delivery is to use fiberoptic probes.

In an experiment performed by the inventors, temperatures were measuredin the brain during an N-TIRE procedure using the Luxtron® m3300Biomedical Lab Kit Fluoroptic® Thermometer (LumaSense™ Technologies,Santa Clara, Calif. USA). STB medical fiber optic probes (LumaSense™Technologies, Santa Clara, Calif. USA) were placed at theelectrode-tissue interface and 7.5 mm along the insulation. FIG. 26A isa photograph showing the N-TIRE electrodes with attached fiber opticprobes, which were used in this intracranial treatment of white matterto measure temperature during pulse delivery.

After insertion of the electrodes, four sets of twenty 50 μs pulses weredelivered with a voltage-to-distance ratio of 1000 V/cm between theelectrodes. The electrode exposure and separation distance were each 5mm. The polarity of the electrodes was alternated between the sets tominimize charge build-up on the electrode surface. These parameters weredetermined from previous in-vivo N-TIRE procedures which showedsufficient ablation of tissue. The NanoKnife® was synchronized with thedog's heart rate in order to prevent any ventricular defibrillation orarrhythmias.

For treatment planning purposes, in order to model accurate N-TIREtreatment, it is beneficial to incorporate changes in conductivity dueto permeabilization of the tissue (as described in detail in thetreatment planning section of this specification), as well asincorporate information relating to temperature changes. See P. A.Garcia, J. H. Rossmeisl, R. E. Neal, II, T. L. Ellis, J. Olson, N.Henao-Guerrero, J. Robertson, and R. V. Davalos, “IntrracranialNon-Thermal Irreversible Electroporation: In vivo analysis,” Journal ofMembrane Biology, p. (in press), 2010, which is incorporated byreference herein in its entirety. Conductivity changes due to thermaleffects could have important implications with a number of differenttreatment parameters, including electrode geometry and pulse parameters(i.e., duration, number, amplitude, and repetition rate, etc.).

FIG. 26B is a graph showing temperature [° C.] distribution during anN-TIRE treatment in the white matter of a canine subject. Moreparticularly, what is shown is the temperature distribution measured bythe probe located at the electrode-tissue interface and 7.5 mm above theinsulation. It is important to note that the starting temperature wasapproximately 33° C. due to the anesthesia effects and this isneuro-protective during brain procedures in general and that the totalpulse delivery took around 300 seconds. For the probe at the interface,four sets of mild increase in temperatures are seen. The probe in theinsulation also shows some very mild increase in temperature that isprobably due to heat conduction from the treatment region.

The changes in the temperature resulting from N-TIRE are less than 0.5°C. and they are not sufficient to generate thermal damage. This confirmsthat any cell death achieved by the procedure was a direct result ofN-TIRE since at the electrode-tissue interface the highest thermaleffects are expected to be achieved. It is also apparent from this datathat it can be assumed in numerical modeling that electricalconductivity changes due to electroporation only and not temperature.

Example VI: Experimental Results of High-Frequency, Polar Pulses forElectroporation of Cells

A chemical reaction technique was performed to fabricate parallel silverelectrodes on glass microscope slides with 100 μm spacing. Briefly, acommercially available mirroring kit was used to deposit pure silveronto the microscope slides (Angel Gilding Stained Glass Ltd, Oak Park,Ill.). A negative thin film photoresist (#146DFR-4, MG Chemicals,Surrey, British Colombia, Canada) was laid on top of the slide andpassed through an office laminator (#4, HeatSeal H212, General BindingCorporation, Lincolnshire, Ill.). A photomask printed at 20k DPI on atransparent film (Output City, Cad/Art Services Inc, Bandon, Oreg.) wasplaced ink side down onto the photoresist, and slides were exposed to UVlight for 45 seconds. After exposure, the slides were placed in a 200 mLbath containing a 10:1 DI water to negative photo developer(#4170-500ML, MG Chemicals, Surrey, British Colombia, Canada). Theslides were placed in a beaker containing DI water to stop thedevelopment process and gently dried using pressurized air. Electrodestructures on the microscope slides were fabricated by removing allsilver not covered by the patterned photoresist. A two part silverremover was included in the mirroring kit used to deposit the silver.The photoresist was then removed by placing the slide in a bath ofacetone.

Microfluidic channels were fabricated using the patterned photoresist ona microscope slide that had not undergone the silvering process. Liquidphase polydimethylsiloxane (PDMS) in a 10:1 ratio of monomers to curingagent (Sylgrad 184, Dow Corning, USA) was degassed under vacuum prior tobeing poured onto the photoresist master and cured for 1 hour at 100° C.After removing the cured PDMS from the mold, fluidic connections to thechannels were punched in the devices using 1.5 mm core borers (HarrisUni-Core, Ted Pella Inc., Redding, Calif.). The PDMS mold was thenbonded over the glass slides containing the patterned electrodes bytreating with air plasma for 2 minutes in a PDC-001 plasma cleaner(Harrick Plasma, Ithaca, N.Y.).

High voltage electrical wires were taped to the glass slide with exposedwire placed in direct contact with the electrical pads. A drop of highpurity silver paint (Structure Probe Inc., West Chester, Pa.) was placedon the pad and allowed to dry for one hour creating a solid electricalconnection. A drop of 5 minute epoxy (Devcon, Danvers, Mass.), used tosecure the electrical connections, was placed on top of each electrodepad and allowed to cure for 24 hours. Pulses were delivered across theelectrodes as described in EXAMPLE 4 prior to the amplification stage.No amplification was needed as the gap between the electrodes was only100 μm. Therefore, the output signal of a function generator (GFG-3015,GW Instek, Taipei, Taiwan)+/−10 V can be used to generate an electricfield capable of inducing electroporation, as shown in FIGS. 27A-B.

Following culture in DMEM-F12 (supplemented with 10% FBS and 1%penicillin streptomycin) MDA-MB-231 cells were resuspended in a PBSsolution 1:1 with Trypan Blue (0.4%). Trypan Blue is a determinant ofcell membrane integrity, and stains electroporated cells blue, whereasnon-electroporated cells remain transparent. Cells at a concentration of10⁶/ml were injected into the microfluidic channel using a syringe. Thefunction generator was triggered by the microcontroller to deliver 80,50 kHz bursts with a width of 1 ms and an amplitude of 500 V/cm. Resultsshown in FIGS. 28A-B, which shows that 60% transfection efficiency wasobtained when starting with cells that are 92% viable. This efficiencyof reversible electroporation could be improved by either increasing thenumber of pulses or the burst width. Additionally, IRE could beperformed by increasing the applied voltage.

Example VII: Alternate Waveforms for Performing High-FrequencyElectroporation

The analytical model for TMP described in the detailed description ofthe invention was utilized to investigate electroporation of a sphericalcell subject to alternative waveforms. As mentioned, the critical TMP(Φ_(cr)) across the plasma membrane required to induce IRE isapproximately 1 V. Belehradek, J., S. Orlowski, L. H. Ramirez, G. Pron,B. Poddevin, and L. M. Mir, Electropermeabilization of Cells in TissuesAssessed by the Qualitative and Quantitative Electroloading ofBleomycin. Biochimica Et Biophysica Acta-Biomembranes, 1994. 1190(1): p.155-163. This threshold is illustrated in FIGS. 15A-C by the dashed,horizontal line on the TMP profiles. Characteristic waveforms of IREwith unipolar pulses and high-frequency IRE with the corresponding TMPdevelopment across the plasma membrane (Φ_(pm)). All results arepresented at the cell pole (θ=0) to show the maximum TMP around thecell. Further, results are only shown for TMP across the plasmamembrane, as the TMP across the nuclear envelope never approached thepermeabilizing threshold. For an electric field of 1500 V/cm, resultsindicate that a unipolar pulse (FIG. 29A), a 250 kHz bipolar burst (FIG.29B), and 250 kHz bipolar burst that includes delays between the pulses(FIG. 29C) are all capable of inducing IRE. However, the time above thethreshold TMP varies between the different cases. The 1500 V/cm unipolarpulse causes the TMP to rise above the critical threshold for IRE (1 V,dashed line). The 1500 V/cm bipolar burst without a delay and with adelay causes the TMP to oscillate around the same critical threshold.This is investigated further in FIG. 30 for center frequencies of 0,100, 250, 500, and 1000 kHz, with the 0 kHz case representing theunipolar pulse, and electric fields of 1000 V/cm and 1500 V/cm. FIG. 30provides a comparison of time above the critical threshold (Φ_(cr)) forIRE at various center frequencies. The burst width of the bipolarwaveform that included delays was twice as long (40 μs) as thecorresponding burst with no delays in order to generate an equivalentpulse on-time (20 μs). The amount of time that the TMP was above thecritical value was normalized by the on-time and converted to apercentage. FIG. 30 illustrates that, for a given frequency, as theelectric field is increased from 1000 V/cm to 1500 V/cm, the percentageof the burst above the critical TMP also increases. At 250 kHz, IRE ispossible during all waveforms, but at 500 kHz, only the waveforms withamplitudes of 1500 V/cm are capable of inducing IRE. As the centerfrequency of the burst increases, the percentage of the burst above thecritical TMP decreases. However, with the inclusion of delays betweenthe pulses, this characteristic dispersion is shifted towards higherfrequencies. At 1 MHz, only the 1500 V/cm waveform with delays cantheoretically cause IRE.

The theoretical model of TMP suggests that IRE should be possible up to1 MHz for an electric field of 1500 V/cm. Including a delay between thepositive and negative pulses comprising the bipolar burst offers atherapeutic advantage in addition to protecting the MOSFETs in the pulsegeneration system from ringing. By not forcing a discharge of the TMPwith an immediate reversal of polarity, the cell is allowed to return tothe resting TMP according to its characteristic time constant. As aresult, the TMP is maintained above the critical voltage required forIRE for a longer amount of time. This metric has been recognized as apotential indicator of treatment outcomes in electroporation basedtherapies with bipolar waveforms. Garcia, P. A., J. H. Rossmeisl, R. E.Neal, T. L. Ellis, J. D. Olson, N. Henao-Guerrero, J. Robertson, and R.V. Davalos, Intracranial Nonthermal Irreversible Electroporation: InVivo Analysis. Journal of Membrane Biology, 2010. 236(1): p. 127-136.

Other potential waveforms for performing high-frequency electroporationare shown in FIGS. 31A-C, which provide characteristic waveforms of IREwith unipolar pulses and high-frequency IRE with the corresponding TMPdevelopment across the plasma membrane (Φ_(pm)). A unipolar pulse withan amplitude of 1500 V/cm is shown for comparison (FIG. 31A). A waveformwithout delays between polarity reversals (FIG. 31B) can maintain apositive TMP throughout the entire treatment if the duration of positivepolarity is tuned to be slightly longer than the duration of negativepolarity. Similarly, for a waveform that includes delays (FIG. 31C), atrain of positive ultra-short pulses could be used to gradually increasethe TMP up to the critical permeabilizing threshold, and a singleultra-short pulse of negative polarity could follow the train withoutcausing the TMP to go negative. In both examples, the ultra-shortnegative going pulse is designed to maintain the predicted benefits ofhigh-frequency electroporation. Namely, it is predicted that thenegative going pulse will prevent action potential generation and stillpermit a degree of capacitive coupling across epithelial layers. FIG. 32is a chart showing an exemplary output from an in vivo treatment of thebrain with high-frequency, bipolar pulses, where the snapshot is takenwithin a single burst.

Example VIII: The Electric Field Distribution During High-FrequencyElectroporation can be Approximated by the Laplace Equation

A 2D axisymmetric FEM representative of a slab of non-infiltrated fatadjacent to dry skin was simulated using COMSOL 4.2 a (Burlington,Mass.). An energized and grounded electrode were modeled as infinitefins (0.5 mm diameter) separated 0.5 cm from the skin-fat interface, fora total spacing of 1 cm. The electric potential distribution within thetissue was obtained by transiently solving

${{- {\nabla \cdot \left( {\sigma{\nabla\Phi}} \right)}} - {\varepsilon_{0}\varepsilon_{r}{\nabla \cdot \left( \frac{\partial{\nabla\Phi}}{\partial t} \right)}}} = 0.$

Additionally, the homogeneous solution was solved according to theLaplace equation:

−∧·(∧ϕ)=0

For the heterogeneous case, the dielectric properties of various tissueswere chosen from data generated by Gabriel et al. available at(http://niremf.ifac.cnr.it/docs/dielectric/home.html). Gabriel, S., R.W. Lau, and C. Gabriel, The dielectric properties of biological tissues:II. Measurements in the frequency range 10 Hz to 20 GHz. Physics inMedicine and Biology, 1996. 41(11): p. 2251-2269. The data wasinterpolated in Mathematica 7 (Wolfram Research, Inc.) in order toestimate the dielectric properties at 1 kHz and 1 MHz. For thehomogeneous case, the electric field distribution is independent of thedielectric properties. The energized and grounded electrodes weresubtracted from the skin and fat subdomains, and treated purely asboundary conditions at 1000 V and 0V, respectively.

FIGS. 33A and 33B show the electric field distribution during a bipolarburst with the frequencies given in Table IV.

TABLE IV Dielectric properties of skin and fat tissue at variousfrequencies Tissue Frequency Property Skin Fat 1 kHz σ [S/m] 0.0001800.0246 ϵ_(r) 1170 20800 1 MHz σ [S/m] 0.0119 0.0267 ϵ_(r) 792 25

From the surface contour map, at 1 kHz, which is representative of a 500us traditional electroporation pulse, the electric field is highlynon-uniform. A majority of the voltage drop occurs within the skinlayer, and the fat layer remains untreated. However, at 1 MHz, which isrepresentative of a 500 ns high-frequency electroporation pulse, thevoltage drop is distributed more uniformly throughout the entire domain.As a result, both the skin and fat layers can be treated. Additionally,the electric field distribution at 1 MHz closely resembles that of thehomogenous solution. Therefore, knowledge of dielectric properties andintricate geometrical arrangements of heterogeneous tissues can beneglected during treatment planning for high-frequency electroporation.This greatly reduces treatment planning protocols and produces morepredictable outcomes.

Example IX

The present invention provides an advancement over tissue ablationtechniques previously devised by providing improved devices and methodsfor precisely and rapidly ablating diseased, damaged, disordered, orotherwise undesirable biological tissues in situ. As used herein, theterm ablation is used to indicate destruction of cells, but notnecessarily destruction of the underlying extracellular matrix. Morespecifically, the present invention provides new devices and methods forablating target tissues for the treatment of diseases and disorders, andparticularly tumors of the brain, using IRE. Use of IRE to decellularizediseased tissue provides a controlled, precise way to destroy aberrantcells of a tissue or organ, such as tumor or cancer cells or masses ofthe brain.

Non-thermal IRE is a method to kill undesirable cells using electricfields in tissue while preserving the ECM, blood vessels, and neuraltubes/myelin sheaths. Certain electrical fields, when applied across acell, have the ability to permeabilize the cell membrane through aprocess that has come to be called “electroporation”. When electricalfields permeabilize the cell membrane temporarily, after which the cellssurvive, the process is known as “reversible electroporation”.Reversible electroporation has become an important tool in biotechnologyand medicine. Other electrical fields can cause the cell membrane tobecome permeabilized, after which the cells die. This deadly process isknown as “irreversible electroporation”. According to the presentinvention, non-thermal irreversible electroporation is a minimallyinvasive surgical technique to ablate undesirable tissue, for example,tumor tissue. The technique is easy to apply, can be monitored andcontrolled, is not affected by local blood flow, and does not requirethe use of adjuvant drugs. The minimally invasive procedure involvesplacing needle-like electrodes into or around the targeted area todeliver a series of short and intense electric pulses that inducestructural changes in the cell membranes that promote cell death. Thevoltages are applied in order to electroporate tissue without inducingsignificant Joule heating that would significantly damage major bloodvessels and the ECM. For a specific tissue type and set of pulseconditions, the primary parameter determining the volume irreversiblyelectroporated is the electric field distribution within the tissue.Recent IRE animal experiments have verified the many beneficial effectsresulting from this special mode of non-thermal cell ablation, such aspreservation of major structures including the extracellular matrix,major blood vessels, and myelin sheaths, no scar formation, as well asits promotion of a beneficial immune response. Due to the nature of thefunction of the brain, in treatment of brain tissues, such as braintumors, the total electrical charge delivered is at least as importantas maintaining low temperature.

In a first aspect, the present invention provides a method for treatingaberrant cell growth in animals. In general, the method comprisesinserting one or more electrodes into or immediately adjacent toaberrant cell masses and applying IRE to cause irreversible cell deathto the aberrant cells. In some embodiments, two or more electrodes areused to treat aberrant cell masses and effect cell death. The electrodesmay be present on the same or different devices. Preferably, theparameters for IRE are selected to minimize or avoid excessive heatingof the treated tissue and surrounding tissue, thus reducing collateraldamage to healthy tissue near the aberrant cell mass. In addition, it ispreferable to minimize the total electrical charge delivered whentreating brain tissue to avoid complications. The methods areparticularly well suited for treatment of aberrant cell growths in or onthe brain, as it is important to avoid collateral damage to brain tissueduring treatments of that organ. The methods also can be applied totreat a number of other of cancers, including liver cancer, prostatecancer, and pancreatic adenocarcinoma.

Viewed differently, the method for treating aberrant cell growth inanimals can be considered a method of treating an animal (includinghumans) having an aberrant cell growth or mass in or on a tissue or anorgan. In exemplary embodiments, the organ is a brain, and the aberrantcell mass is a benign or malignant tumor. Under this view, the methodcan be a method of treating an animal suffering from a disease ordisorder resulting from aberrant cell growth by reducing or eliminatingsome or all of a mass (e.g., tumor) produced by the aberrant cellgrowth.

To effect the methods according to the invention, the present inventionprovides devices designed to treat aberrant cell masses usingirreversible electroporation (IRE). While IRE devices have beendisclosed prior to the priority date of this document, advanced surgicaltools for in vivo IRE to treat diseased tissues and organs had not beendeveloped. The present invention, for the first time, provides devicessuitable for in vivo IRE treatment of diseases and disorders,particularly those associated with abnormal cell growth in or on atissue or organ, which allow for minimally invasive treatment ofpatients suffering from such abnormal cell growth. The present inventorshave designed microsurgical tools to treat currently inoperable tumorsin humans and other animals through IRE, and in particular brain tumors.While not so limited, the designs provided herein are sufficient toablate the majority of tumors smaller than about 3 cm in diameter, suchas those about 14 cc in volume or less.

The present invention extends and improves on prior techniques for IREby providing new methods and devices for IRE treatment of solid tumors,including those associated with brain cancer. Because the brain issusceptible to small fluctuations in temperature, the present inventionprovides devices and techniques for non-thermal IRE to kill undesirablecells and tissues. In addition, because the brain functions by way ofelectrical charges, the present invention provides devices andtechniques that limit or precisely control the amount of electricalcharge delivered to tissue. To achieve the invention, a device has beendeveloped that contains both conducting and non-conducting surfaces andthat is capable of delivering controlled pulses of electricity to tumortissues while substantially protecting surrounding healthy tissue. Inexemplary embodiments, the device has a laminate structure of at leastone electrically conductive and at least one electrically insulativematerial. In some exemplary embodiments, the device has at least twoconcentric disk electrodes separated by an insulating material similarin dimensions to those already used in deep brain stimulation (DBS). DBSis an FDA approved therapy that alleviates the symptoms of otherwisetreatment-resistant disorders, such as chronic pain, Parkinson'sdisease, tremor, and dystonia. The Examples, below, present resultsdemonstrating that an IRE procedure does not induce substantial thermaleffects in the brain, and delivers electrical charges to highly definedregions of tissues, supporting the conclusion that IRE can be used as aminimally invasive surgical technique for the treatment of brain cancerand other diseases and disorders involving aberrant cell massdevelopment. The methods employ the unique designs discussed herein,which provide improved controlled delivery of electrical pulses withcontrolled three-dimensional patterns and controlled thermal outputs.The present devices and systems provide surgical tools and methods forIRE treatment of subcutaneous tumors that expand the application spacefor this new technology, with the potential to treat a number ofcancers, including brain, liver, prostate and pancreatic adenocarcinoma.

In practicing the method, the number of electrodes, either on a singleor multiple devices, used can be selected by the practitioner based onthe size and shape of the tumor to be treated and the size and shape ofthe electrode. Thus, embodiments of the invention include the use ofone, two, three, four, five, or more electrodes. Each electrode can beindependently sized, shaped, and positioned in or adjacent the tumor tobe treated. In addition, the number and spacing of electrodes on asingle device can be adjusted as desired. As detailed below, thelocation, shape, and size of electrodes can be selected to producethree-dimensional killing zones of numerous shapes and sizes, allowingfor non-thermal treatment of tumors of varying shapes and sizes.

Surprisingly, it has been found that pulse durations for ablation ofsolid tumors can be relatively short, thus reducing the probability ofgeneration of thermal conditions and excessive charges that causecollateral damage to healthy tissues. More specifically, the presentinvention recognizes for the first time that, in contrast to priordisclosures relating to IRE, the pulse length for highly efficienttissue ablation can be lower than 100 microseconds (100 us). Indeed, ithas surprisingly been determined that a pulse length of 25 us or lowercan successfully cause non-thermal cell death. Thus, in embodiments, themethod of treatment uses pulse lengths of 10 us, 15 us, 20 us, 25 us, 30us, 35 us, 40 us, 45 us, 50 us, 55 us, 60 us, 65 us, 70 us, 75 us, 80us, 85 us, or 90 us. Preferably, to most effectively minimize peripheraldamage due to heat, pulse lengths are limited to 90 us or less, forexample 50 us or less, such as 25 us. By reducing the pulse length, ascompared to prior art techniques for IRE, larger electric fields can beapplied to the treatment area while avoiding thermal damage tonon-target tissue (as well as to target tissue). As a result of thedecreased pulse length and concomitant reduction in heat production, themethods of the invention allow for treatment of tissues having highervolumes (e.g., larger tumors) than possible if prior art methods were tobe employed for in vivo treatment of tumors.

It has also been determined that voltages traditionally used for IRE aretoo high for beneficial treatment of tumors in situ. For example,typically, IRE is performed using voltages of between 4000 V/cm to 1500V/cm. The present invention provides for use of voltages of much lowerpower. For example, the present methods can be performed using less than1500 V/cm. Experiments performed by the inventors have shown that 2000V/cm can cause excessive edema and stroke in patients when applied tobrain tissue. Advantageously, for treatment of brain tumors, appliedfields of about 500 V/cm to 1000 V/cm are used. Thus, in general fortreatment of brain tumors, applied fields of less than 1000 V/cm can beused.

Further, it has been discovered that the number of electrical pulsesthat can be applied to successfully treat tumors can be quite high.Prior art methods of using IRE for various purposes included the use ofrelatively few pulses, for example 8 pulses or so. Reports of use of upto 80 pulses for IRE have been published; however, to the inventors'knowledge, a higher number of pulses has not been recommended. Thepresent invention provides for the use of a relatively high number ofpulses, on the order of 90 pulses or greater. For example, in exemplaryembodiments, 90 pulses are used. Other embodiments include the use ofmore than 90 pulses, such as 100 pulses, 110 pulses, or more.

According to the method of the invention, cycle times for pulses are setgenerally about 1 Hz. Furthermore, it has been found that alternatingpolarity of adjacent electrodes minimizes charge build up and provides amore uniform treatment zone. More specifically, in experiments performedby the inventors, a superficial focal ablative IRE lesion was created inthe cranial aspect of the temporal lobe (ectosylvian gyrus) using theNanoKnifeB (Angiodynamics, Queensbury, N.Y.) generator, blunt tipbipolar electrode (Angiodynamics, No. 204002XX) by delivering 9 sets often 50 us pulses (voltage-to-distance ratio 2000 V/cm) with alternatingpolarity between the sets to prevent charge build-up on the stainlesssteel electrode surfaces. These parameters were determined from ex-vivoexperiments on canine brain and they ensured that the charge deliveredduring the procedure was lower than the charge delivered to the humanbrain during electroconvulsive therapy (an FDA approved treatment formajor depression). Excessive charge delivery to the brain can inducememory loss, and thus is preferably avoided.

The method of the invention encompasses the use of multiple electrodesand different voltages applied for each electrode to precisely controlthe three-dimensional shape of the electric field for tissue ablation.More specifically, it has been found that varying the amount ofelectrical energy emitted by different electrodes placed in a tissue tobe treated allows the practitioner to finely tune the three-dimensionalshape of the electrical field that irreversibly disrupts cell membranes,causing cell death. Likewise, the polarity of electrodes can be variedto achieve different three-dimensional electrical fields. Furthermore,one of the advantages of embodiments of the invention is to generateelectric field distributions that match complex tumor shapes bymanipulating the potentials of multiple electrodes. In theseembodiments, multiple electrodes are energized with different potentialcombinations, as opposed to an “on/off” system like radio frequencyablation, to maximize tumor treatment and minimize damage to surroundinghealthy tissue.

The method of the invention is implemented using devices and systems.The devices according to the invention are suitable for minimallyinvasive temporary implantation into a patient, emission of atissue-ablating level of electricity, and removal from the patient. Thedevice according to the invention thus may be used in the treatment oftumors and the treatment of patients suffering from tumors. The devicescan take multiple forms, based on the desired three-dimensional shape ofthe electrical field for cell killing. However, in general, the devicesinclude two or more regions of differing conductivity. In someembodiments, the device comprises alternating regions of conductivity,for example a region of electrical conductivity, which is adjacent aregion of electrical non-conductivity, which is adjacent a differentregion of conductivity. In embodiments, the device comprises two or morelayers of conductive and insulative materials, in a laminate structurewith alternating conductive properties. To protect tissue that is not tobe treated, the outer layer can be insulative except at the region wheretreatment is to be effected. According to embodiments of the device, theamount of conductive material exposed to the tissue to be treated can beadjusted by a movable non-conductive element disposed on the outersurface of the device.

Further, in general, the device takes a rod-like shape, with onedimension (i.e., length) being substantially longer than the other(i.e., width or diameter). While exemplary embodiments are configured ina generally cylindrical shape, it is to be understood that thecross-sectional shape of the electrode can take any suitable geometricshape. It thus may be circular, square, rectangular, oval, elliptical,triangular, pentagonal, hexagonal, octagonal, etc.

The devices of the invention comprise one or more electrodes, which areelectrically conductive portions of the device. The devices are thuselectrically conductive elements suitable for temporary implantationinto living tissue that are capable of delivering an electrical pulse tothe living tissue. The device of the invention has a proximal end and adistal end. The proximal end is defined as the end at which the deviceis attached to one or more other elements, for control of the functionof the device. The distal end is defined by the end that contacts targettissue and delivers electrical pulses to the tissue. The distal end thuscomprises an exposed or exposable electrically conductive material forimplantation into a target tissue. Typically, the distal end isdescribed as including a “tip” to denote the region of the distal endfrom which an electrical pulse is delivered to a tissue. The devicefurther comprises at least one surface defining the length andcircumference of the device.

The device of the invention comprises an electrode tip at the distalend. The electrode tip functions to deliver electrical pulses to targettissue. The tip may be represented by a single conductive layer of thedevice or may be represented by two or more conductive layers that areexposed to the tissue to be treated. Furthermore, the tip may bedesigned to have any number of geometrical shapes. Exemplary embodimentsinclude tips having a needle-like shape (i.e., electrical pulses emanatefrom a small cone-like structure at the distal end of the device) orhaving a circular shape (i.e., electrical pulses emanate from thecylindrical outer surface of the device, which is a section of thedevice where the outer insulative layer has been removed to expose thenext layer, which is conductive). For use in treatment of brain tumors,the tip advantageously comprises a blunt or rounded end to minimizelaceration of brain tissue. In embodiments, the rounded or blunt endcomprises a hole that allows for a sharp or needle-like structure to bedeployed into tumor tissue at the appropriate time.

The device comprises a proximal end, which generally functions forattachment of the device to a power source/controller and a handle. Theproximal end thus may comprise connections for electrical wires that runfrom the power source/controller to the electrically conductive layersof the device. Standard electrical connections may be used to connectthe conductive elements to the wires. In embodiments, the device isattached to a handle for ease of use by a human. While not limited inthe means for attaching the device to the handle, in embodiments, theconnection is made by way of a friction fit between the outer surface ofthe device and the handle, for example by way of an insulative O-ring(e.g., a Norprene O-ring) on the handle. In other embodiments, thedevice comprises, on its outer surface, ridges or other surface featuresthat mate with surface features present on the handle. In yet otherembodiments, the proximal end comprises one or more structures thatallow for controlled movement of the outer surface along the length ofthe device. In such embodiments, the outer surface will comprise anouter sheath that is electrically non-conductive, and which surrounds anelectrically conductive layer. Using the structures at the proximal end,the outer sheath may be moved, relative to the rest of the device, toexpose or conceal varying portions of the electrically conductivematerial beneath it. In this way, the amount of surface area of theconductive material at the tip can be adjusted to provide a desiredheight of exposure of tissue to the electrode tip. Of course, otherstructures for securely fastening the device to a holder may be used,such as clips, set screws, pins, and the like. The device is not limitedby the type of structure used to connect the device to the holder.

The device of the invention can be designed to have any desired size.Typically, it is designed to be minimally invasive yet at the same timesuitable for delivery of an effective electrical field for IRE. Thediameter or width is thus on the order of 0.5 mm to 1 cm. Preferably,the diameter or width is about 0.5 mm to about 5 mm, such as about 1 mm,2 mm, 3 mm, or 4 mm. The length of the device is not particularlylimited, but is generally set such that a surgeon can use the devicecomfortably to treat tumors at any position in the body. Thus, for humanuse, the device is typically on the order of 40 cm or less in length,such as about 30 cm, 25 cm, or 15 cm, whereas for veterinary use, thelength can be much larger, depending on the size of animal to betreated. For treatment of human brain tumors, the length can be on theorder of 40 cm.

In some embodiments, the device, or a portion of it, is flexible. Aflexible device is advantageous for use in accessing tumorsnon-invasively or minimally invasively through natural body cavities. Inembodiments where the device or a portion of it is flexible, the shapeof the device can change based on contact with body tissues, can bepre-set, or can be altered in real-time through use of wires or othercontrol elements, as known in the art, for example in use withlaparoscopic instruments.

The device of the invention can be part of a system. In addition to thedevice, the system can comprise a handle into or onto which the deviceis disposed. The handle can take any of a number of shapes, but isgenerally designed to allow a surgeon to use the device of the inventionto treat a patient in need. It thus typically has a connector forconnecting the device to the holder, and a structure for the surgeon tograsp and maneuver the device. The handle further can comprise a triggeror other mechanism that allows the surgeon to control delivery ofelectrical pulses to the device, and thus to the tissue to be treated.The trigger can be a simple on/off switch or can comprise a variablecontrol that allows for control of the amount of power to be deliveredto the device. Additionally, the handle may be created in such a mannerthat it may be attached to additional pieces of equipment, such as onesthat allow precise placement of the electrode relative to an inertial orthe patient's frame of reference, allowing steady and accurate electrodepositioning throughout an entire procedure, which may entail theapplication of electric pulses in addition to radiotherapy, imaging, andinjections (systemically and locally) of bioactive agents. Furthermore,the handle may be attached to machines that are operated remotely bypractitioners (e.g., the Da Vinci machine)

The system can further comprise a power source and/or a power controlunit. In embodiments, the power source and control unit are the sameobject. The power source provides electrical power to the device,typically by way of an electrical connection through the handle. Thepower source can be any suitable source that can deliver the properamount of electrical power to the device of the invention. Suitablepower sources are commercially available, and the invention is notlimited by the type or manufacturer. The power control unit provides theuser with the ability to set the power output and pulse time forelectrical pulses to be delivered to the device, and thus to the tissueto be treated. Suitable control units are commercially available, andthe invention is not limited by the type or manufacturer. For example,an appropriate power source/controller is available from Angiodynamics(Queensbury, N.Y.).

Example X: IRE Performance Indicia

To illustrate 1) the possibility to monitor creation of a cell-freetissue section in brain in real-time using imaging techniques, 2) thevariety of tissues that can be used, and 3) how to preserve vasculature,a healthy female purpose bred beagle was used. Nine sets of ten pulseswere delivered with alternating polarity between the sets to minimizecharge build-up on the electrode surfaces. The maximumvoltage-to-distance ratio used was 2000 V/cm because the resultingcurrent did not exceed 2 amps. The charge that was delivered to thebrain during the IRE procedure was 22.5 mC, assuming ninety pulses (50us pulse durations) that would result from a maximum hypotheticalcurrent of 5 amps.

TABLE V IRE pulse parameters EXPOSURE GAP VOLTAGE TO PULSE LENGTHDISTANCE VOLTAGE DISTANCE DURATION ELECTRODES [mm] [mm] [V] RATIO [V/cm]PULSES [μs] 1 mm 5 5  500 1000 90 50 Monopolar Bipolar Standard 7 16002000 90 50

Method: After induction of general anesthesia, a routine parietotemporalcraniectomy defect was created to expose the right temporal lobe of thebrain. Two decelluarization sites were performed: 1) a deep lesionwithin the caudal aspect of the temporal lobe using a monopolarelectrode configuration (6 mm electrode insertion depth perpendicular tothe surface of the target gyrus, with 5 mm interelectrode distance), and2) a superficial lesion in the cranial aspect of the temporal lobe usinga bipolar electrode (inserted 2 cm parallel to the rostrocaudal lengthof the target gyrus, and 2 mm below the external surface of the gyrus).Intraoperative adverse effects that were encountered included grossmicrohemorrhages around the sharp monopolar electrode needles followinginsertion into the gyrus. This hemorrhage was controlled with topicalapplication of hemostatic foam. Subject motion was completelyobliterated prior to ablating the superficial site by escalating thedose of atracurium to 0.4 mg/kg. Grossly visible brain edema and surfaceblanching of the gyrus overlying the bipolar electrode decelluarizationsite was apparent within 2 minutes of completion of IRE at this site.This edema resolved completely following intravenous administration of1.0 g/kg of 20% mannitol. No adverse clinically apparent effectsattributable to the IRE procedure, or significant deterioration inneurologic disability or coma scale scores from baseline evaluationswere observed. However, the results indicated to the inventors that alower voltage would provide adequate results but with less ancillarytrauma to the brain.

Methods to monitor creation of cell-free tissues in vivo: A uniqueadvantage of IRE to ablate tissues in vivo is its ability to bemonitored in real-time using imaging techniques, such as electricalimpedance tomography, MRI, and ultrasound. Below, this Example shows MRIexaminations performed immediate post-operatively, which demonstratethat IRE decelluarization zones were sharply demarcated (FIGS. 34A-C).

As shown in FIGS. 34A-C neurosonography performed intraoperatively andat 1 hour and 24 hours post-procedure demonstrated clearly demarcateddecellularization zones and visible needle tracts within the targetedbrain parenchyma. Intraoperatively and immediately postoperatively, thedecellularization zones appeared as hypoechoic foci with needle tractsappearing as distinct hyperechoic regions (FIG. 35 ).Neurosonographically, at the 24 hour examination the IREdecellularization zone was hypoechoic with a hyperechoic rim (FIG. 35 ).Compared to the 1 hour post-operative sonogram, the IRE decelluarizationzone appeared slightly larger (1-2 mm increase in maximal, twodimensional diameter). EEG performed in the post-operative periodrevealed focal slowing of the background rhythm over the right temporalregion in association with the decelluarization zones.

Macrolevel and histologic verification of treating cells: The brain wascollected within 2 hours of the time of death and removed from thecranium. Care was taken to inspect soft tissues and areas of closurecreated at the time of surgery. The brain was placed in 10% neutralbuffered formalin solution for a minimum of 48 hours. Then, the brainwas sectioned at 3 mm intervals across the short axis of the brain, inorder to preserve symmetry and to compare lesions. Following grossdissection of fixed tissues, photographs were taken of brain sections inorder to document the position and character of lesions, as shown inFIG. 36 . Readily apparent in gross photographs of the sectioned brainare lesions created either by the physical penetration of brainsubstance with electrodes or created by the application of pulse throughthe electrodes. There are relatively well-demarcated zones of hemorrhageand malacia at the sites of pulse delivery.

Microscopic lesions correlated well with macroscale appearance. Areas oftreatment are represented by foci of malacia and dissociation of whiteand grey matter. Small perivascular hemorrhages are present and there issparing of major blood vessels (see FIG. 37B). Notable in multiplesections is a relatively sharp line of demarcation (approximately 20-30micrometers) between areas of frank malacia and more normal, organizedbrain substance (see FIG. 37A).

Analysis to determine IRE threshold: To determine the electric fieldneeded to irreversibly electroporate tissue, one can correlate thelesion size that was observed in the ultrasound and MRI images with thatin the histopathological analysis to determine the percentage of lesiongrowth. Decellularized site volumes can be determined afteridentification and demarcation of IRE decellularization zones fromsurrounding brain tissue using hand-drawn regions of interest (ROI). Arepresentative source sample image is provided in FIG. 38 .

Example XI: Modeling of Electrode Shape and Placement

The present invention provides simple and elegant minimally invasivemicrosurgical tools to treat currently inoperable tumors in humans andanimals through IRE. Exemplary designs are shown in FIGS. 39-43 .

FIG. 39A depicts an example of a device 700 according to one embodimentof the invention. This embodiment is fully compatible with existingelectroporation electronics and comprises a surgical probe/electrode tip710 at its distal end, which includes both ground electrodes 711 andenergized electrodes 712. The device further comprises a universalconnector 750 at its proximal end. The device also comprises internalwiring 770 to deliver electrical impulses to the tip 710. The body ofthe device is defined by surface 718.

The size and shape of the IRE area is dictated by the voltage appliedand the electrode configuration and is readily predictable throughnumerical modeling. Therefore, different surgical tips can be fashionedto achieve the same therapeutic result. For example, tip 710 cancomprises retractable conductive spikes 713 emanating from a blunt endtip 710 and disposed, when deployed, at an acute angle to tip 710 (seeFIG. 39B). Alternatively, tip 710 can be fashioned as a point or needle,and can include retractable accordion-type conductive elements 714 (seeFIG. 39C). In other exemplary embodiments, tip 710 can comprise multipleretractable spikes 715 that, when deployed, emanate at 90° C. from tip710 (see FIG. 39D). Yet again, tip 710 can comprise retractableconductive spikes 716 emanating from a needle-end tip 710 and disposed,when deployed, at an acute angle to tip 710 (see FIG. 39E). FIGS. 39B,39D, and 39E show probes with parallel circular channels 717 ofapproximately 1 mm that protrude through the length of the electrodeholder. Each channel has the capability of guiding individual 1 mmelectrodes to the treatment area. Towards the bottom of the holder, thechannels deviate from their straight path at a specific angle. Theelectrodes can be Platinum/Iridium with an insulating polyurethanejacket to ensure biocompatibility, similar to materials that are used inDBS implants. Different protrusion depths of the electrodes within thetissue as well as the applied voltage can be used to control the size ofthe treated area.

The devices can comprise interchangeable surgical tips, allowing forversatility in creating a device well suited for different tissues ordifferent sized or shaped tumors. Varying electrode diameters (varied inpart by selection of the type and length of deployable spikes) andseparation distances will be sufficient to ablate the majority of tumorsabout or smaller than 3 cm by selecting the appropriate voltages tomatch different tumor sizes and shapes. As shown in later figures, someof the embodiments of the device comprise an element at the tip tointroduce anti-cancer drugs for ECT, cytotoxic proteins, or otherbioactive agents into the targeted area.

While not depicted in detail, embodiments of the device comprise durablecarbon coatings over portions of the device that act both to insulatenormal tissue and to increase the efficiency of IRE pulsing.

With general reference to FIG. 39A-D in brain tumor IRE treatment, forexample, a single blunt-end device with embedded active and groundelectrodes can be used. In an embodiment not particularly depicted inthe figures, the device contains a primary blunt-end tip with a holedisposed in the end, for insertion through delicate, soft brain tissue.The device of these embodiments further comprises a secondary sharp tip,which can be deployed through the hole in the blunt-end primary tip,which allows for penetration into the tumor tissue, which can besubstantially more dense or hard, and not easily punctured by ablunt-end tip. In general, the device of the invention is typicallysimilar in dimensions (2 mm) to those already used in deep brainstimulation (DBS), which ensures that they are feasible for surgicalapplications. DBS uses electrodes in an FDA approved therapy toalleviate the symptoms of otherwise treatment-resistant disorders, suchas Parkinson's disease and dystonia. Furthermore, the electrodepositioning frame, which is used in stereotactic surgery in conjunctionwith imaging systems, can be used to position the surgical probes andensure that the position of the electrodes is optimal. Simulations of adesign similar to the one in FIG. 39A show treatment volumes comparableto typical brain tumors.

Turning now to FIGS. 40A-C, the figures depict in more detail anembodiment of a tip 810 according to the invention. FIG. 40A depicts anexploded view of tip 810, showing multiple concentric layers ofconducting 820 and non-conducting 830 materials. An outer layer orsheath 860 of non-conducting material is shown with perforations 861. Anouter, perforated layer 832 is disposed around the concentric rings ofmaterials, to allow for delivery of bioactive substances to cells inproximity to the device when in use. Perforated layer 832 may bedisposed in full, direct contact with the outermost layer of theconcentric ring structure, or may be substantially separated from thering structure by chamber 833 that holds cooling fluid.

As shown in the cut-away depiction in FIG. 40B, device tip 810 hasmultiple alternating layers of conducting 820 and non-conducting 830materials surrounding an non-conducting inner core 831. In FIG. 40B, thetop and bottom conducting regions 820 are energized electrodes while themiddle conducting region 820 is a ground electrode. The presentinvention provides the conducting and non-conducting (insulative)regions in varying lengths to fine tune electrical field generation.More specifically, using imaging techniques directed at the tumor to betreated, a surgeon can determine what type of electrical field is bestsuited for the tumor size and shape. The device can comprise one or moremovable elements on the surface of the tip (not depicted) or can bedesigned such that one or more of the alternating conducting 820 ornon-conducting 830 elements is movable. Through movement and setting ofthe outer element(s) or inner elements 820 or 830, the surgeon canconfigure the device to deliver a three-dimensional electrical killingfield to suit the needs of the particular situation.

FIG. 40C depicts the concentric laminate structure of tip 810, viewedfrom the distal end along the distal-proximal axis, showing again thelaminate nature of the device.

In addition to changing charges, adapting the physical dimensions of theprobe also allows flexibility in tailoring the treatment area to matchthe dimensions of the tumor. By altering the electrode parameters,including diameter, length, separation distance, and type, it ispossible to conveniently tailor the treatment to affect only specific,targeted regions. In addition, developing an electrode capable ofaltering and adapting to these dimensional demands greatly enhances itsusability and adaptability to treatment region demands.

Example XII: Hollow Core Device

Many IRE treatments may involve coupled procedures, incorporatingseveral discrete aspects during the same treatment. One embodiment ofthe invention provides a device with a needle-like tip 910 with anincorporated hollow needle 990 with either an end outlet 991 (shown inFIG. 41A) or mixed dispersion regions 961 (shown in FIG. 41B). Such aconfiguration allows for highly accurate distribution of injectablesolutions, including those comprising bioactive agents. Use of such adevice limits the dose of treatment required as well as ensures thecorrect placement of the materials prior to, during, and/or after thetreatment. Some of the possible treatment enhancers that would benefitfrom this technology are: single or multi-walled carbon nanotubes(CNTs); chemotherapeutic agents; conductive gels to homogenize theelectric field; antibiotics; anti-inflammatories; anaesthetics; musclerelaxers; nerve relaxers; or any other substance of interest.

The schematics in FIGS. 41A-B show two basic hollow needle designs thatmay be implemented to enhance solution delivery prior to, during, orafter IRE treatment. They both have multiple conducting surfaces 920that may act as charged electrodes, grounded electrodes, or electricresistors, depending on the treatment protocol. FIG. 41A shows a hollowtip 910 for injection of agents at its end while FIG. 41B hasdistributed pores 961 throughout for a more generalized agentdistribution. As shown in FIG. 41B, the pores are disposed in thenon-conducting regions 930 of the device.

Irreversible Electroporation (IRE), a new minimally invasive techniquewe invented to treat tumors, can be enhanced using carbon nanotubes(CNTs). The technique can be used on a variety of tumors includingliver, prostate, pancreatic adenocarcinoma and renal carcinoma. Focalablation techniques, such as IRE, however, are not selective and thuscannot distinguish between healthy and cancerous cells. To overcome thislimitation, nanoparticles can be incorporated into IRE therapy.Nanomaterials offer a potential means for energy focusing, because theypresent a toolset with a unique size range closely matching that ofcells (1 to 1,000 nm), and substantial multi-functional capability. Someembodiments of nanoparticles exhibit a “lightning rod” effect whenexposed to electric fields, amplifying the field at the nanoparticle'stip, thereby producing a significantly larger electric potentialcompared to its surroundings and reducing the possibility of sub-lethaljoule heating. This localized amplification of electric fields couldthus be used as a means to induce IRE from relatively small electricfields; residual adverse effects to surrounding tissue wouldsubsequently be reduced. Targeting of nanoparticles through tumorspecific antibodies to the desired tissue region will allow treatment upto and beyond the tumor margin using IRE, and offer the opportunity tolower the IRE applied field, thereby minimizing damage to surrounding,non-cancerous tissue during treatment. Integration of CNTs into IREcould more selectively localize the electric field and thermal profileto cancer cells through antibody targeting and more precisely controlthe induction of cell death and HSP expression.

When carbon nanotubes (CNTs) are immersed in an electric field, aninduced dipole is generated that tends to align the axis of the CNTparallel to the electric field. Taking advantage of these effects can beused to reduce cell damage during treatment. For example, two sets ofelectric fields delivered subsequent to and at right angles to eachother is a technique that can be used to align the CNTs andelectroporate the cells. Under some circumstances, cells electroporatedusing CNTs may result in cells having a higher vitality than whenelectroporated without the use of CNTs. The use of CNTs injected into aregion of tissue, with or without targeting antibodies, to mediate IREfor tumor ablation is another method covered by the present invention.

In N-TIRE therapy, the local electric field distribution dictates thetreatment area. When electric field parameters are optimized, N-TIREpossesses a clear therapeutic advantage in that there is no induction ofthermal injury in the ablated area, thereby preserving important tissuecomponents such as the extracellular matrix, major blood vessels, myelinsheaths, and nerves. Since N-TIRE is a focal ablation technique, it doesnot selectively kill infiltrative cancer cells with the potential forre-growth and metastasis beyond the tumor margin without affecting thesurrounding healthy cells. The ablation area can be enlarged withoutinducing joule heating and the selectivity of N-TIRE can be enhancedthrough the use of CNTs. Localized amplification of electric fields fromCNTs could induce N-TIRE in adjacent cells from relatively smallelectric fields, without affecting healthy surrounding cells. Further,antibody targeting of CNTs to tumor cells could permit localizedCNT-mediated electric field amplification at selected tumor cellmembranes causing targeted cell death due to permanent membranedestabilization. Even further, it is advantageous to incorporate CNTsinto N TIRE protocols in order to simultaneously lower the voltage forN-TIRE and expand the treatable area.

Combinatorial CNT-mediated N-TIRE cancer therapies can include treatmentof a number of cancers including prostate, liver, kidney, andpancreatic. Breast cancer is a particularly apt application since thiscombinatorial therapy can directly address the need of scar reductionand mitigate the likelihood of metastasis, which have proven in somecircumstances to be helpful for improved treatment. Adapting N-TIREtreatments for breast carcinomas has several unique challenges. Amongthese are the diverse and dynamic physical and electrical properties ofbreast tissue. The fatty and connective tissues within the breast regionsurrounding a tumor have low water content, and thus significantlyreduced electrical conductivity and permittivity than tumors. It hasbeen shown that N-TIRE treatment area is highly predictable based onelectric field distribution. CNTs will provide a means to raise theelectric field magnitude within the tumor and increase N-TIRE treatmentarea in localized breast carcinomas.

Selective destruction of tumor cells with CNT-mediated N-TIRE therapy isdependent upon targeting CNTs to the tumor cells of interest. Inphysiological conditions, cells uptake folic acid across the plasmamembrane using the folate carrier to supply the folate requirements ofmost normal cells. In contrast, folate receptor (FR), a high affinitymembrane folate-binding protein, is frequently overexpressed in a widevariety of cancer cells. Since it is generally either absent or presentat only low levels in most normal cells, the FR has been identified asnot only a marker of cancers but also a potential and attractive targetfor tumor-specific drug delivery. Thus, bioconjugated nanoparticles,such as those conjugated with folic acid (FA-NP), can be synthesized andused as drug delivery tools for administering drugs into cancer cells.

Example XIII: Devices Comprising Active Cooling

In embodiments, the device comprises a cooling system within theelectrode to reduce the highly localized temperature changes that occurfrom Joule heating. During the electric pulses for IRE, the highestquantity of heat generation is at the electrode-tissue interface. Byactively cooling (for example, via water flow) the electrode during theprocedure, these effects are minimized. Further, cooling provides a heatsink for the nearby tissue, further reducing thermal effects. Thisallows more flexibility in treating larger tissue regions with IRE whilekeeping thermal effects negligible, providing a greater advantage forIRE over conventional thermal techniques. Cooling can be achieved byplacement of one or more hollow chambers within the body of the device.The cooling chambers can be closed or open. Open chambers can beattached at the proximal end to fluid pumping elements to allow forcirculation of the fluid (e.g., water) through the device during use.

Example XIV: Movable Outer Sheath

In embodiments, the device comprises an outer protector that is designedto be movable up and down along the length of the device. FIG. 10depicts such a movable outer protector. More specifically, FIG. 10depicts a device 1000 comprising tip 1010 that includes outer protector1062 that can be moved up and down along the length of device 1000. Inpractice, outer protector 1062 is disposed fully or partially encasingouter sheath 1060. After or during insertion into tissue to be treated,outer protector 1062 is retracted partially to expose outer sheath 1060,which in the embodiment depicted comprises mixed dispersion outlets1061. As such, the number of dispersion outlets 1061 exposed to thetissue during treatment can be adjusted to deliver varying amounts ofbioactive agent to different portions of the tissue being treated. Anymechanism for movement of the outer sheath along the device may be used.In embodiments, screw threads are disposed on the upper portion of thedevice, allowing for easy adjustment by simple twisting of the outersheath. Alternatively, set screws may be disposed in the outer sheath,allowing for locking of the sheath in place after adjustment.

Example XV: System for IRE Treatment of Tumors

The invention provides a system for performing IRE tumor tissueablation. As depicted in FIG. 11 , an exemplary system can comprise adevice 1100 reversibly attached to holder 1140. Holder 1140 can comprisetrigger 1141, which allows the user to control the flow of electricityfrom power source/controller 1142 to device 1100.

In this embodiment, device 1100 comprises further elements for use. Morespecifically, device 1100 comprises a height adjustment apparatus 1151at its proximal end to effect movement of outer sheath 1160. Outersheath 1160 further comprises markings or scores 1168 on its surface toindicate amount of movement of outer sheath 1160 after implantation ofdevice 1100 into tumor tissue.

Example XVI: Use of Multiple Electrode Charges

We have discovered that a highly customizable electric fielddistribution may be attained by combining multiple electrode chargeswithin the same pulse. This allows a highly customized and controllabletreatment protocol to match the dimensions of the target tissue. Inaddition, the invasiveness of the treatment may be decreased by reducingthe number of electrode placements required for treatment. In order todemonstrate the great flexibility in electric field distribution shape,2-dimensional and axis symmetric models were developed with 3 and 4electrode arrays along a single axis. The results are depicted in FIGS.44A-D. For development of the data, only the electric potentials of theelectrodes were manipulated to achieve the great flexibility needed inIRE treatment planning. For FIGS. 44A and 44B, four charged electrodesof alternating polarity at 2500V and ground were used to develop a 2-Dreadout (FIG. 44A) and axis symmetric electrode configurations (FIG.44B). Four charged electrodes with the center two at 5000V and 0V andthe outer two electrodes at 2500V were used to develop a 2-D readout(FIG. 44C) and axis symmetric electrode configurations (FIG. 44D). Threecharged electrodes with the center one at 2500V and the outer two at 0Vwere used for 2-D (FIG. 44E) and axis symmetric electrode (FIG. 44F)configurations. Three charged electrodes with the center at 0V, leftelectrode at 5000V, and right electrode at 2500V for 2-D (FIG. 44G) andaxis symmetric (FIG. 44H) scenarios. Three charged electrodes with thecenter at 1750V, left electrode at 3000V and right electrode at 0V for2-D (FIG. 44I) and axis symmetric electrode (FIG. 44J) configurations.

Example XVII: Altering the Diameter and Shape of Electrodes

We have done some preliminary studies and determined that the electricfield distribution may be altered, and thus controlled, by changing thediameter and shape of the electrode between the conducting surfaces.This fact can be used to design and develop an electrode with anexpandable/contractible interior and deformable exterior to change itssize in real-time before or during a treatment to alter, and thusspecify the electric field distribution in a manner that may bedesirable during treatment. The ability to adjust this dimension inreal-time is made additionally useful by the fact that a significantlysmaller electrode may be inserted to keep it minimally invasive, andthen expand the dimension once the electrode has reached the targettissue. In embodiments, the invention includes the use of a balloonbetween regions of charge that may be inflated/deflated during treatmentto alter field distribution. FIGS. 45A-C, depict modeling of a bulgingregion between the charges in a bipolar electrode. Three differentmodels that study the inclusion of a balloon between the two electrodesin a bipolar design are shown. FIG. 45A (861.21 mm³ treated area) has noballoon for comparison purposes. The middle design of Panel B (795.71mm³ treated area) has an elongated balloon that is in close proximity tothe electrodes. The bottom design of Panel C (846.79 mm³ treated area)has a smaller balloon that helps distribute the electric field.

Example XVIII: Alternating Polarity

With the application of electric potentials, electrical forces may driveions towards one electrode or the other. This may also lead toundesirable behavior such as electrolysis, separating water into itshydrogen and oxygen components, and leading to the formation of bubblesat the electrode-tissue interface. These effects are further exacerbatedfor multiple pulse applications. Such effects may cause interferencewith treatment by skewing electric field distributions and alteringtreatment outcomes in a relatively unpredictable manner. By altering thepolarity between the electrodes for each pulse, these effects can besignificantly reduced, enhancing treatment predictability, and thus,outcome. This alternating polarity may be a change in potentialdirection for each pulse, or occur within each pulse itself (switch eachelectrode's polarity for every pulse or go immediately from positive tonegative potential within the pulse at each electrode).

Example XIX: Bipolar and Monopolar Electrodes

Using a bipolar electrode with 4 embedded electrodes, one can use themiddle two electrodes to inject a sinusoidal current (^(˜)5 mA) that islow enough in magnitude to not generate electroporation and measure thevoltage drop across the remaining two electrodes. From this setup onecan calculate the impedance of the tissue and gather the conductivity ofthe tissue which is needed for treatment planning. One can do thisanalysis in a dynamic form after each electroporation pulse.Conductivity increases as a function of temperature and electroporation;therefore, for accurate treatment predictions and planning, the dynamicconductivity is needed and we can use the bipolar or unipolar electrodesto map the conductivity distribution before IRE treatment and during toadjust the pulse parameters.

Example XX: Parameters

The following are parameters that can be manipulated within the IREtreatments discussed herein.

-   -   Pulse length: 5 us-1 ms    -   Number of pulses: 1-10,000 pulses    -   Electric Field Distribution: 50-5,000 V/cm    -   Frequency of Pulse Application: 0.001-100 Hz    -   Frequency of pulse signal: 0-100 MHz    -   Pulse shape: square, exponential decay, sawtooth, sinusoidal,        alternating polarity    -   Positive, negative, and neutral electrode charge pulses        (changing polarity within probe)    -   Multiple sets of pulse parameters for a single treatment        (changing any of the above parameters within the same treatment        to specialize outcome)    -   Electrode type        -   Parallel plate: 0.1 mm-10 cm diameter        -   Needle electrode(s): 0.001 mm-1 cm diameter        -   Single probe with embedded disk electrodes: 0.001 mm-1 cm            diameter        -   Spherical electrodes: 0.0001 mm-1 cm diameter    -   Needle diameter: 0.001 mm-1 cm    -   Electrode length (needle): 0.1 mm to 30 cm    -   Electrode separation: 0.1 mm to 5 cm

The present invention has been described with reference to particularembodiments having various features. It will be apparent to thoseskilled in the art that various modifications and variations can be madein the practice of the present invention without departing from thescope or spirit of the invention. One skilled in the art will recognizethat these features may be used singularly or in any combination basedon the requirements and specifications of a given application or design.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention. It is intended that the specification and examples beconsidered as exemplary in nature and that variations that do not departfrom the essence of the invention are intended to be within the scope ofthe invention.

1-29. (canceled)
 30. A method comprising: operatively coupling a firstmonopolar electrode and a second monopolar electrode to a generator,wherein the first monopolar electrode comprises an active distal tip;inserting the active distal tip of the first monopolar electrode into atarget tissue; activating the generator to apply biphasic electricalpulses between the first monopolar electrode and the second monopolarelectrode in an amount sufficient to non-thermally ablate cells in thetarget tissue, wherein the biphasic electrical pulses comprise a voltageof up to 5,000 volts and a frequency in the range of 200 kHz-100 MHz.31. The method of claim 30, wherein the biphasic electrical pulses areconfigured to maintain a temperature of the target tissue at 60° C. orless.
 32. The method of claim 30, wherein the first monopolar electrodefurther comprises a lumen, the active distal tip further comprises aneedle shape and the electrode has a length in the range of 0.1 mm to 30cm.
 33. The method of claim 30, wherein an outer surface of the firstmonopolar electrode is at least partially covered by a non-conductivematerial and the active distal tip comprises a conductive material. 34.The method of claim 30, wherein the biphasic electrical pulses comprisea voltage of up to 3,000 volts.
 35. The method of claim 30, wherein thetarget tissue comprises soft tissue, prostate tissue, liver tissue,kidney tissue, pancreatic tissue, breast tissue, or brain tissue. 36.The method of claim 30, wherein the active distal tip comprises either asingle conductive layer or at least two conductive layers, the firstmonopolar electrode comprises a diameter between 0.001 millimeter to 1centimeter, and the electrode has a length of 0.1 millimeter to 30centimeters.
 37. The method of claim 30, wherein the step of activatingthe generator to apply biphasic electrical pulses between the firstmonopolar electrode and the second monopolar electrode in an amountsufficient to non-thermally ablate cells in the target tissue isconfigured to promote a beneficial immune response in the target tissue.38. The method of claim 30, further comprising the step of operativelycoupling the first monopolar electrode to a remotely controllableapparatus.
 39. The method of claim 30, wherein the biphasic electricalpulses comprise multiple sets of pulse parameters for a singletreatment.
 40. The method of claim 39, wherein the multiple sets ofpulse parameters are configured to result in different electric fielddistributions within the target tissue.
 41. The method of claim 39,wherein the step of activating the generator to apply biphasicelectrical pulses between the first monopolar electrode and the secondmonopolar electrode in an amount sufficient to non-thermally ablatecells in the target tissue is configured to reduce a likelihood ofelectrolysis, or a formation of bubbles at an interface of the monopolarelectrode.
 42. A method comprising: inserting an expandable electrodeinto a target tissue, wherein the expandable electrode is operativelycoupled to a generator; expanding the expandable electrode from aconstricted state to an expanded state; activating the generator toapply biphasic electrical pulses from the expandable electrode in anamount sufficient to non-thermally ablate cells in the target tissue,wherein the biphasic electrical pulses comprise a voltage of up to 5,000volts and a frequency in the range of 200 kHz-100 MHz.
 43. The method ofclaim 42, wherein the step of activating the generator to apply biphasicelectrical pulses from the expandable electrode in an amount sufficientto non-thermally ablate cells in the target tissue is configured toreduce a likelihood of electrolysis, or a formation of bubbles at aninterface of the monopolar electrode.
 44. The method of claim 42,further comprising the step of: monitoring an impedance of the targettissue in real-time.
 45. The method of claim 42, further comprising thestep of: mapping an electrical conductivity of the target tissue. 46.The method of claim 42, wherein the biphasic electrical pulses comprisemultiple sets of pulse parameters for a single treatment and themultiple sets of pulse parameters are configured to result in differentelectric field distributions within the target tissue.
 47. A methodcomprising, inserting an electrode into a target tissue, wherein theelectrode is operatively coupled to a generator; mapping an electricalconductivity of the target tissue; activating the generator to applybiphasic electrical pulses from the electrode in an amount sufficient tonon-thermally ablate cells in the target tissue; and monitoring animpedance of the target tissue in real-time.
 48. The method of claim 47,wherein the step of monitoring the impedance is configured to be usedduring a treatment prediction or a treatment planning.
 49. The step ofclaim 47, further comprising the step of: adjusting one or more pulseparameters.